REVIEW URRENT C OPINION
Haemophilus influenzae: recent advances in the understanding of molecular pathogenesis and polymicrobial infections Farshid Jalalvand and Kristian Riesbeck
Purpose of review Non-typeable Haemophilus influenzae (NTHi) is a human-specific mucosal pathogen and one of the most common causes of bacterial infections in children and patients with chronic obstructive pulmonary disease. It is also frequently found in polymicrobial superinfections. Great strides have recently been made in the understanding of the molecular mechanisms underlying NTHi pathogenesis. Recent findings By using new methodology, such as experimental human colonization models and whole-genome approaches, investigators have shed light upon the various strategies of NTHi that are involved in pathogenesis. These include the escape of the mucociliary elevator, evasion of host immunity, survival in environments with scarce nutrients, and finally participation in polymicrobial infections. Lipooligosaccharide branching, proteinous adhesins, metabolic adaption to nutrient availability and many scavenging systems are implicated in these processes. Interestingly, genome-based studies comparing virulent and commensal strains have identified many hypothetical proteins as virulence determinants, suggesting that much regarding the molecular pathogenesis of NTHi remains to be solved. Summary NTHi is an opportunistic pathogen and highly specialized colonizer of the human respiratory tract that has developed intricate mechanisms to establish growth and survival in the human host. Continued research is needed to further elucidate NTHi host–pathogen and pathogen–pathogen interactions. Keywords Haemophilus influenzae, immune evasion, molecular pathogenesis, niche adaptation, polymicrobial infections
INTRODUCTION Haemophilus influenzae is a commensalistic Gramnegative species of the human upper respiratory tract microbiota and a frequent cause of mucosal infections [1]. As a model organism, H. influenzae has been involved in several major scientific achievements throughout history; it was the source of the isolation of the first restriction enzyme [2], it was used to develop the first animal model for bacterial meningitis [3] and was also the first free-living organism to have its genome fully sequenced [4]. Furthermore, the first licensed glycoconjugate vaccine for use in humans was developed against encapsulated H. influenzae type b (Hib) [5], and, more recently, H. influenzaederived protein D has successfully been employed as the carrier protein for a licensed pneumococcal conjugate vaccine [6]. www.co-infectiousdiseases.com
Non-typeable, that is, uncapsulated H. influenzae (NTHi) is a prevalent cause of acute and recurrent otitis media in children and exacerbations in chronic obstructive pulmonary disease (COPD) patients [7–11]. It is also the etiological agent of bacterial pulmonary infections in immunocompromised hosts such as lung cancer patients, and occasionally disseminates to cause invasive disease [11–14]. To establish a successful colonization, Medical Microbiology, Department of Laboratory Medicine Malmo¨, Lund University, Malmo¨, Sweden Correspondence to Dr Kristian Riesbeck, Medical Microbiology, Department of Laboratory Medicine Malmo¨, Lund University, Jan Waldenstro¨ms gata 59, SE-205 02 Malmo¨, Sweden. Tel: +46 40 338494; e-mail: [email protected] Curr Opin Infect Dis 2014, 27:268–274 DOI:10.1097/QCO.0000000000000056 Volume 27 Number 3 June 2014
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Haemophilus influenzae pathogenesis Jalalvand and Riesbeck
KEY POINTS NTHi causes mucosal and invasive opportunistic infections in humans to a high socioeconomic cost. NTHi is associated with a large degree of genetic heterogeneity and interstrain DNA exchange. NTHi is highly adapted to colonize the human respiratory tract and evades swift clearance by the host immune system via virulence factors including lipo-oligosaccharide, proteinous adhesins and binding of complement inhibitors. NTHi has developed intricate systems to metabolically respond to changes in the microenvironment. Recent studies have elucidated some of the molecular mechanisms underlying polymicrobial infections involving NTHi and co-pathogens, and these include viral attenuation of the host immune response.
NTHi has to cope with the structural barrier of the mucociliary epithelium, the innate and acquired immunity, intermicrobial competition, and nutrient poor environments. NTHi is highly adapted to these conditions and usually establishes a transient colonization with a turnover rate of 3 months; the host is overtaken by a new strain as the immune system clears the preceding one [15]. As NTHi currently causes the vast majority of H. influenzae infections, this review is primarily focused on recent advances related to the molecular pathogenesis of NTHi.
GENETIC INTRASPECIES HETEROGENEITY H. influenzae is a genetically diverse species with each strain containing between 1765 and 2355 genes (http://www.ncbi.nlm.nih.gov/genome/genomes/ 165). The core-genome present in all strains consists of 1485 genes, or about 75% of the genomic content of any given isolate [16 ]. The supragenome of H. influenzae, however, has been predicted to contain approximately 4500 unique genes. As many strains are naturally competent, it is plausible that a constant and dynamic genetic exchange occurs via uptake and recombination of intraspecies DNA containing unique H. influenzae uptake sequences [17], with individual strains having access to various parts of the supragenome during concomitant colonization of the same host [18 ,19]. This mechanism would allow the bacteria to access a wide variety of genes while still keeping their individual genomes small, thus giving them a fitness advantage. Specific disease-associated genetic elements, as opposed to asymptomatic colonization factors, have &&
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long remained elusive, although some genes have been found to be more prevalent among virulent strains, as has been reviewed [20]. However, when the complete gene content of 210 geographically and clinically diverse NTHi strains was recently compared, 149 genes were identified to be significantly associated with either virulence or commensalism [16 ]. Interestingly, the 28 genes that were more likely to be found in virulent strains were not any of the well characterized virulence factors involved in adherence, lipo oligosaccharide (LOS) biosynthesis, or immune evasion. Indeed, most were hypothetical proteins indicating that the principal virulence determinants may have eluded researchers thus far. &&
HOST COLONIZATION AND PERSISTENCE The initial step of successful colonization and subsequent infection is adherence to the host tissue. To circumvent mechanical clearance by the mucosal epithelium, NTHi uses four main strategies: perturbance of the mucociliary elevator, adherence to the epithelium, formation of microcolonies and biofilm, and/or invasion of epithelial cells. One mechanism underlying NTHi-mediated decreased ciliary beating and detachment of ciliated cells was recently shown to involve the activation of host protein kinase C epsilon [21]. However, disintegrity of ciliated cells is not always observed, as reported by other investigators using extended time co-culture (1–10 days) with NTHi and primary human respiratory epithelial cells [22]. Instead, bacteria appeared to seek refuge in paracellular foci to evade the mucociliary elevator. Intercellular bacteria were seemingly associated with the basal cell layers, where junctional disorganization was observed. Both these strategies can plausibly be implemented during human colonization. Epithelial cells are connected to the structural scaffold of the underlying extracellular matrix (ECM) via a range of basal surface structures including integrins [23]. Should these interactions be disrupted (as observed in the studies above), or the epithelium be damaged by viral infections or chronic inflammation, ECM proteins become a viable target for adherence by pathogens [24]. Like other respiratory tract pathogens, NTHi has evolved strategies to bind to ECM proteins. We recently reported that NTHi protein F, a homolog of streptococcal laminin-binding proteins Lbp/Lmb, is a novel adhesin that directly mediates bacterial binding to laminin and primary human bronchial epithelial cells [25 ]. These results are in analogy with NTHi adhesins protein E and Hap that have previously been reported to interact with both ECM proteins as
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well as host cells [26–28]. Interestingly, protein F (gene also annotated as hfeA) has been found to be crucial for successful NTHi infection in vivo [29 ]. Animal models with mice, rats, and chinchillas have traditionally been used to mimic human NTHi infections, but the relevance of the data obtained in these studies will always remain an issue because of the human-specific nature of the pathogen. In a new and exciting study, Winokur et al. [30 ] developed a human nasopharyngeal colonization model to study the virulence of NTHi in its natural host. Thirteen selected phase-variable genes were monitored during a 6-day period of human colonization and significant changes were observed in two genes, phosphorylcholine kinase licA and IgA1-protease igaB [31 ]. LicA incorporates exogenously acquired phosphorylcholine into LOS, an alteration that mediates increased adherence and entry into bronchial epithelial cells. The in-vivo selection of subpopulations shifting to phase-on expression of licA suggests that this mechanism is of importance during early colonization. Invasion and persistence of NTHi in epithelial cells have been extensively reviewed [32]. There is also increasing evidence showing that NTHi may reside in lymphoid tissues and act as a reservoir for recurrent infections [33,34]. Additional mechanisms of H. influenzae persistence are reported to involve classic toxin–antitoxin systems that may arrest cell proliferation during stressful conditions via mRNA targeting and result in dormant persister cells [35]. In a recent report, NTHi IgA1 proteases were implicated in internalization and intracellular persistence in bronchial epithelial cells [36]. IgA1 protease is a secreted virulence factor with high specificity for the predominant mucosal immunoglobulin, secretory IgA1, which it cleaves in the hinge region to circumvent the host humoral response. As a testament to its importance for pathogenesis, the protease was one of two phase-variable genes shown to be significantly upregulated during experimental human colonization [31 ]. Two variants of IgA1 protease have been identified in NTHi, IgaA and, more recently, IgaB [36]. Authors found that IgaA was required for optimal invasion of epithelial cells, whereas IgaB was needed for optimal intracellular persistence. Intriguingly, it is unclear how IgaA enhances NTHi internalization. Possible mechanisms could be a yet unidentified enzymatic function that the secreted protease exerts on bacterial and/or host cells, or interaction between the outer membrane-integrated residual IgaA translocator domain and epithelial cells. Albeit previously controversial, the formation of NTHi biofilm in vivo is nowadays generally acknowledged by most researchers. NTHi residing in &&
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biofilms, as compared with planktonic subsistence, acquire a series of advantages including enhanced resistance to antimicrobial compounds and host immune effectors [37,38 ]. The current understanding of NTHi biofilms has recently been excellently reviewed in several publications [37,38 ,39,40]. &&
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NICHE ADAPTABILITY AND NUTRIENT ACQUISITION The human nasopharynx is a nutrient-poor environment for bacteria, especially during inflammation when the host sequesters essential nutrients [41 ]. To establish a successful colonization of the respiratory tract, otopathogens rely on their ability to adapt to this niche, scavenge scarce nutrients, and respond metabolically to changes in the environment. A recent study found that H. influenzae carbonic anhydrase is essential for growth in low CO2 concentrations and important for prolonged intracellular survival in phagosomes, suggesting a role for this enzyme in bacterial adaption to different CO2 conditions and acidic pH [42]. The pathogen also utilizes urease, found to be more prevalent in disease-causing isolates than commensals, to survive in acid environments [43,44]. Other systems involved in sensing the microenvironment within the host include the multifunctional Sap-transporter, possibly via detection of host molecules as well as heme accessibility [45,46]. As alluded to by its name, Haemophilus lacks the ability to synthesize heme that is essential for its survival. Heme is thus acquired exogenously and the pathogen has developed many ways for sensing extracellular heme (and iron) and altering its gene expression to correctly respond to the availability of the metabolite [47,48]. Expectedly, disruption of these systems results in decreased virulence [46–48]. One of the central transcriptional regulators involved in iron-acquisition is the ferric uptake regulator (Fur) protein. The Fur regulon of NTHi was recently defined and shown to contain 73 genes [49]. Interestingly, authors found that in addition to iron uptake and utilization genes, IgA1 protease was also regulated by Fur, suggesting that the bacterium has coupled low iron concentrations to the mucosal milieu. An isogenic Dfur mutant was found to be attenuated in vivo. Fur is not the only regulatory responder to low iron concentrations. Recently, the core modulon (identical in five different isolates) of H. influenzae adaptation to low levels of iron/heme was defined in vitro and verified in vivo [50 ]. In addition to the 55 genes that were similarly regulated in all strains, authors identified approximately 200 non-core &
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Haemophilus influenzae pathogenesis Jalalvand and Riesbeck
genes that they hypothesize are important for environmental adaptation. The conditions in the lower respiratory tract of COPD patients differ significantly from those in the nasopharynx, with mucus hypersecretion (that probably causes hypoxic microenvironments), failure of cilia-mediated clearance, high cytokine levels, excessive phagocyte presence, epithelial disintegrity, and oxidant/antioxidant imbalances [51]. The genetic adaptation of NTHi populations to these changes have been examined and 15 genetic islands have been identified to be more prevalent in COPD isolates as compared with commensal nasopharyngeal strains, including genes encoding the IgA1 protease, high-molecular-weight adhesins, and urease [52]. Interestingly, some of these genes had previously been observed to be transcriptionally upregulated during bacterial growth in sputum obtained from COPD patients, suggesting in-situ selection for both genetically and transcriptionally adapted strains [53]. Other factors that potentially contribute to the etiology of exacerbations in COPD patients include bacterial oxidative stress responses and impaired host defenses targeting NTHi [53,54]. The topic of reactive oxygen species-resisting NTHi mechanisms has been thoroughly reviewed recently [55 ]. &
INFLAMMATION AND IMMUNE EVASION In recent years, great achievements have been made in the molecular understanding of NTHi-mediated inflammation processes during otitis media and COPD exacerbations [56,57]. The pathogenesis differs in these conditions, with the balance being shifted toward immune suppression and immune activation in otitis media and COPD, respectively. Nevertheless, evading the host innate immunity, particularly the complement system, is essential for all human bacterial pathogens [58]. NTHi relies on two main strategies for complement evasion: camouflage using branched LOS that covers the bacterial surface or binding of host complement regulator proteins. By attaching a variety of compounds to the LOS core, including scavenged host-specific sialic acid and phosphorylcholine, NTHi blocks the accessibility of bactericidal antibodies to its other surface structures, thus inhibiting complement activation via the classical pathway [59,60 ,61 ,62,63]. Interestingly, this mechanism has shown to be induced in vivo and during exposure to human serum [31 ,60 ,61 ,62]. Other host– pathogen interactions that LOS has been implicated in include biofilm formation and resistance to antimicrobial peptides [64 ]. We have previously shown that acquisition of the host complement inhibitor vitronectin is &
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important for NTHi serum resistance. By using a proteomic approach to identify vitronectin-binding NTHi proteins, we found that Haemophilus protein F mediates complement evasion via sequestration of vitronectin at the bacterial surface [65 ]. &
POLYMICROBIAL INFECTIONS Recent insight into bacterial cell–cell interactions has shown stunning displays of specific and speciesunique secretory responses to direct stimuli [66 ]. It is therefore not surprising that mixed pneumococcal/NTHi otitis media are suggested to be clinically distinct infections compared with single organism otitis media [67]. Co-colonization/infections have long been observed with the otopathogens NTHi, pneumococci, and Moraxella catarrhalis [68–74], but some studies have also reported antagonistic relationships between them [75,76]. Moreover, secondary NTHi infections are commonly detected in patients infected by viral pathogens such as influenza A virus (IAV), rhinovirus, and human respiratory syncytial virus [71,77–80]. It could be that NTHi is particularly well adapted to interplay with other bacteria as well as to host microenvironments affected by primary viral infections. The understanding of the molecular mechanisms underlying polymicrobial superinfections has improved during the last years. One such mechanism was recently unfolded as investigators showed that rhinovirus-induced degradation of the host signaling pathway adaptor protein IRAK-1 attenuates the TLR2-mediated response to NTHi [81 ]. This results in delayed neutrophil recruitment that hinders swift bacterial clearance, providing one explanation as to why rhinoviral/NTHi superinfections are frequently seen clinically. We recently shed light on the physiological interplay between NTHi and group A streptococci (GAS), two pathogens whose presence has been shown to correlate in children suffering from acute pharyngotonsillitis [82,83 ]. We showed that NTHiderived outer membrane vesicles (OMVs) carried functional b-lactamase that protected GAS from amoxicillin-mediated killing. Interestingly, NTHi OMV shedding is seen in several infection models, suggesting that they may play important roles during pathogenesis [22,45]. In a seminal article published by Wong et al. [29 ], investigators employed transposon insertion sequencing to define the NTHi genes needed during single organism infection, co-infection with IAV, and the core set of genes required during both conditions. In addition to observing that IAV made mice highly susceptible to NTHi infections, authors noted that several (but not all) bacterial genes
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involved in defense against various stress conditions were dispensable in co-infections, suggesting that IAV (like rhinovirus) attenuates the host immune response to NTHi. Other interesting findings included the bacterial adaptation to the changes in nutrient access brought on by the viral infection, including alterations in stress responses. These data provide valuable insight in to the idiosyncrasies of polymicrobial infections, and the continued elucidation of the dynamic molecular interactions augmenting NTHi disease in polymicrobial settings will be highly interesting to follow.
Conflicts of interest There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
CONCLUSION NTHi is a remarkable pathogen in many aspects. In contrast to many of its co-pathogens, NTHi does not have any known cytotoxic effectors or exotoxins, secreted proteolytic enzymes (aside from the IgA1specific endopeptidases), lipases, super antigens, injectisome secretion systems, or a polysaccharide capsule. Still it manages to efficiently outcompete other microbes and colonize the upper respiratory tract of children and the lower respiratory tract of immunocompromised adults. How does it do it? NTHi has developed advanced and efficient scavenging systems for obtaining nutrients in the human airways. Indeed, it has been reported that the pathogen produces siderophore-utilization proteins without producing siderophores [84], meaning it steals iron from other bacterial species that in turn steal it from the host! Furthermore, NTHi scavenges essential and host-specific nutrients, effectively saving the cost of synthesizing these products. The price of the loss of biosynthesis enzymes is niche dependency, as NTHi can only sustain prolonged growth in the human respiratory tract. We have gained increasing insight into other virulence mechanisms of NTHi. Investigators have shown that NTHi employs an array of multifunctional proteins, including protein D, protein E, protein F, the Sap-transporter and the heme-binding proteins HbpA and TehB, a display of highly economic use of proteins [25 ,26,27,45,46,65 ,85– 89]. Moreover, genetic heterogeneity and accessibility to a diverse supragenome has provided NTHi with additional fitness benefits as each isolate can maintain a relatively small genome and still be adaptable to environmental selection [16 ]. To summarize, NTHi is a highly adapted human-specific commensal whose molecular pathogenesis is not yet fully understood. The clinical burden of this opportunistic pathogen encourages more research to help combat infections caused by it. &
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Acknowledgements This work was supported by grants from the Alfred O¨sterlund, the Anna and Edwin Berger, Greta and Johan Kock foundations, the Swedish Medical Research Council (grant number 521-2010-4221, www.vr.se), the Physiographical Society (Forssman’s Foundation), and Ska˚ne County Council’s research and development foundation.
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Proc Natl Acad Sci U S A 2013; 110:15413–15418. This article identifies the essential NTHi genes required for single and co-infection with influenza A virus in a murine model. 30. Winokur PL, Chaloner K, Doern GV, et al. Safety and immunological out&& comes following human inoculation with nontypeable Haemophilus influenzae. J Infect Dis 2013; 208:728–738. This article shows developement of a human colonization model for NTHi. 31. Poole J, Foster E, Chaloner K, et al. Analysis of nontypeable Haemophilus && influenzae phase-variable genes during experimental human nasopharyngeal colonization. J Infect Dis 2013; 208:720–727. The results from experimental human coloninzation by NTHi show the relative importance of a selected group of phase-variable genes. 32. Clementi CF, Murphy TF. Nontypeable Haemophilus influenzae invasion and persistence in the human respiratory tract. Front Cell Infect Microbiol 2011; 1:1. 33. Stepinska M, Olszewska-Sosinska O, Lau-Dworak M, et al. 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Novel concepts in nontypeable Haemophilus && influenzae biofilm formation. FEMS Microbiol Lett 2013; 346:81–89. This is the review summing up the current understanding of NTHi biofilm formation. 39. Swords WE. Quorum signaling and sensing by nontypeable Haemophilus influenzae. Front Cell Infect Microbiol 2012; 2:100. 40. Swords WE. Nontypeable Haemophilus influenzae biofilms: role in chronic airway infections. Front Cell Infect Microbiol 2012; 2:97. 41. Krismer B, Liebeke M, Janek D, et al. Nutrient limitation governs Staphylo& coccus aureus metabolism and niche adaptation in the human nose. PLoS Pathog 2014; 10:e1003862. This article shows the identification of nutrient availability in the nasopharynx and subsequent development of a synthetic nasal medium supporting bacterial growth. 42. Langereis JD, Zomer A, Stunnenberg HG, et al. Nontypeable Haemophilus influenzae carbonic anhydrase is important for environmental and intracellular survival. J Bacteriol 2013; 195:2737–2746. 43. 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46. Vogel AR, Szelestey BR, Raffel FK, et al. SapF-mediated heme-iron utilization enhances persistence and coordinates biofilm architecture of Haemophilus. Front Cell Infect Microbiol 2012; 2:42. 47. Steele KH, O’Connor LH, Burpo N, et al. Characterization of a ferrous ironresponsive two-component system in nontypeable Haemophilus influenzae. J Bacteriol 2012; 194:6162–6173. 48. Hempel RJ, Morton DJ, Seale TW, et al. The role of the RNA chaperone Hfq in Haemophilus influenzae pathogenesis. BMC Microbiol 2013; 13:134. 49. Harrison A, Santana EA, Szelestey BR, et al. Ferric uptake regulator and its role in the pathogenesis of nontypeable Haemophilus influenzae. Infect Immun 2013; 81:1221–1233. 50. Whitby PW, Vanwagoner TM, Seale TW, et al. Comparison of transcription of & the Haemophilus influenzae iron/heme modulon genes in vitro and in vivo in the chinchilla middle ear. BMC Genomics 2013; 14:925. This is an interesting insight into the iron/heme-limitation modulon of NTHi. 51. 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This is an extensive review of how NTHi copes with exogenous and endogenous oxidative stress. 56. Liu K, Chen L, Kaur R, Pichichero ME. Transcriptome signature in young children with acute otitis media due to nontypeable Haemophilus influenzae. Int Immunol 2013; 25:353–361. 57. Rotta Detto Loria J, Rohmann K, Droemann D, et al. Haemophilus influenzae infection upregulates the NLRP3 inflammasome and leads to caspase-1dependent secretion of interleukin-1beta: a possible pathway of exacerbations in COPD. PLoS One 2013; 8:e66818. 58. Zipfel PF, Hallstrom T, Riesbeck K. Human complement control and complement evasion by pathogenic microbes – tipping the balance. Mol Immunol 2013; 56:152–160. 59. Clark SE, Snow J, Li J, et al. Phosphorylcholine allows for evasion of bactericidal antibody by Haemophilus influenzae. PLoS Pathog 2012; 8:e1002521. 60. Clark SE, Eichelberger KR, Weiser JN. Evasion of killing by human antibody & and complement through multiple variations in the surface oligosaccharide of Haemophilus influenzae. Mol Microbiol 2013; 88:603–618. This article describes the elucidation of the molecular mechanisms behind LOSmediated complement evasion. 61. Langereis JD, Stol K, Schweda EK, et al. Modified lipooligosaccharide & structure protects nontypeable Haemophilus influenzae from IgM-mediated complement killing in experimental otitis media. MBio 2012; 3:e00079-12. This is another article that reveals the molecular mechanisms behind LOSmediated complement evasion. 62. Nakamura S, Shchepetov M, Dalia AB, et al. Molecular basis of increased serum resistance among pulmonary isolates of nontypeable Haemophilus influenzae. PLoS Pathog 2011; 7:e1001247. 63. Apicella MA. Nontypeable Haemophilus influenzae: the role of N-acetyl-5neuraminic acid in biology. Front Cell Infect Microbiol 2012; 2:19. 64. Morey P, Viadas C, Euba B, et al. Relative contributions of lipooligosaccharide && inner and outer core modifications to nontypeable Haemophilus influenzae pathogenesis. Infect Immun 2013; 81:4100–4111. This is a systematic evaluation of the role of various components of LOS in NTHi pathogenesis. 65. Su YC, Jalalvand F, Morgelin M, et al. Haemophilus influenzae acquires & vitronectin via the ubiquitous Protein F to subvert host innate immunity. Mol Microbiol 2013; 87:1245–1266. This article describes the elucidation of the molecular mechanisms behind protein F-mediated complement evasion. 66. Traxler MF, Watrous JD, Alexandrov T, et al. Interspecies interactions stimulate && diversification of the Streptomyces coelicolor secreted metabolome. MBio 2013; 4:e00459-13. This article provides an interesting insight into the bacterial species-specific metabolic change upon cell–cell interaction. 67. Dagan R, Leibovitz E, Greenberg D, et al. Mixed pneumococcal-nontypeable Haemophilus influenzae otitis media is a distinct clinical entity with unique epidemiologic characteristics and pneumococcal serotype distribution. J Infect Dis 2013; 208:1152–1160. 68. Shiri T, Nunes MC, Adrian PV, et al. Interrelationship of Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus colonization within and between pneumococcal-vaccine naive mother-child dyads. BMC Infect Dis 2013; 13:483.
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Pathogenesis and immune response 69. Hong W, Khampang P, Erbe C, et al. Nontypeable Haemophilus influenzae inhibits autolysis and fratricide of Streptococcus pneumoniae in vitro. Microbes Infect 2013. [Epub ahead of print] 70. Ruohola A, Pettigrew MM, Lindholm L, et al. Bacterial and viral interactions within the nasopharynx contribute to the risk of acute otitis media. J Infect 2013; 66:247–254. 71. van den Bergh MR, Biesbroek G, Rossen JW, et al. Associations between pathogens in the upper respiratory tract of young children: interplay between viruses and bacteria. PLoS One 2012; 7:e47711. 72. Tikhomirova A, Kidd SP. Haemophilus influenzae and Streptococcus pneumoniae: living together in a biofilm. Pathog Dis 2013; 69:114–126. 73. Brockson ME, Novotny LA, Jurcisek JA, et al. Respiratory syncytial virus promotes Moraxella catarrhalis-induced ascending experimental otitis media. PLoS One 2012; 7:e40088. 74. Verhaegh SJ, Snippe ML, Levy F, et al. Colonization of healthy children by Moraxella catarrhalis is characterized by genotype heterogeneity, virulence gene diversity and co-colonization with Haemophilus influenzae. Microbiology 2011; 157:169–178. 75. Xu Q, Almudervar A, Casey JR, Pichichero ME. Nasopharyngeal bacterial interactions in children. Emerg Infect Dis 2012; 18:1738–1745. 76. Dahlblom V, Soderstrom M. Bacterial interactions in the nasopharynx – effects of host factors in children attending day-care centers. J Infect Public Health 2012; 5:133–139. 77. Molyneaux PL, Mallia P, Cox MJ, et al. Outgrowth of the bacterial airway microbiome after rhinovirus exacerbation of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2013; 188:1224–1231. 78. Blyth CC, Webb SA, Kok J, et al. The impact of bacterial and viral co-infection in severe influenza. Influenza Other Respir Viruses 2013; 7:168–176. 79. Hishiki H, Ishiwada N, Fukasawa C, et al. Incidence of bacterial coinfection with respiratory syncytial virus bronchopulmonary infection in pediatric inpatients. J Infect Chemother 2011; 17:87–90. 80. Arrevillaga G, Gaona J, Sanchez C, et al. Respiratory syncytial virus persistence in macrophages downregulates intercellular adhesion molecule-1 expression and reduces adhesion of nontypeable Haemophilus influenzae. Intervirology 2012; 55:442–450.
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81. Unger BL, Faris AN, Ganesan S, et al. Rhinovirus attenuates nontypeable Haemophilus influenzae-stimulated IL-8 responses via TLR2-dependent degradation of IRAK-1. PLoS Pathog 2012; 8:e1002969. This is an elucidation of the molecular mechanisms in rhinoviral attenuation of the host response to NTHi. 82. Brook I, Gober AE. Increased recovery of Moraxella catarrhalis and Haemophilus influenzae in association with group A beta-haemolytic streptococci in healthy children and those with pharyngo-tonsillitis. J Med Microbiol 2006; 55:989–992. 83. Schaar V, Uddback I, Nordstrom T, Riesbeck K. Group A streptococci are & protected from amoxicillin-mediated killing by vesicles containing beta-lactamase derived from Haemophilus influenzae. J Antimicrob Chemother 2014; 69:117–120. This article shows bacterial polymicrobial collaboration related to antibiotic pressure. 84. Morton DJ, Turman EJ, Hensley PD, et al. Identification of a siderophore utilization locus in nontypeable Haemophilus influenzae. BMC Microbiol 2010; 10:113. 85. Johnson RW, McGillivary G, Denoel P, et al. Abrogation of nontypeable Haemophilus influenzae protein D function reduces phosphorylcholine decoration, adherence to airway epithelial cells, and fitness in a chinchilla model of otitis media. Vaccine 2011; 29:1211–1221. 86. Mason KM, Raffel FK, Ray WC, Bakaletz LO. Heme utilization by nontypeable Haemophilus influenzae is essential and dependent on Sap transporter function. J Bacteriol 2011; 193:2527–2535. 87. Shelton CL, Raffel FK, Beatty WL, et al. Sap transporter mediated import and subsequent degradation of antimicrobial peptides in Haemophilus. PLoS Pathog 2011; 7:e1002360. 88. Vergauwen B, Elegheert J, Dansercoer A, et al. Glutathione import in Haemophilus influenzae Rd is primed by the periplasmic hemebinding protein HbpA. Proc Natl Acad Sci U S A 2010; 107:13270– 13275. 89. Whitby PW, Seale TW, Morton DJ, et al. Characterization of the Haemophilus influenzae tehB gene and its role in virulence. Microbiology 2010; 156: 1188–1200.
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Haemophilus influenzae: recent advances in the understanding of molecular pathogenesis and polymicrobial infections Farshid Jalalvand and Kristian Riesbeck
Purpose of review Non-typeable Haemophilus influenzae (NTHi) is a human-specific mucosal pathogen and one of the most common causes of bacterial infections in children and patients with chronic obstructive pulmonary disease. It is also frequently found in polymicrobial superinfections. Great strides have recently been made in the understanding of the molecular mechanisms underlying NTHi pathogenesis. Recent findings By using new methodology, such as experimental human colonization models and whole-genome approaches, investigators have shed light upon the various strategies of NTHi that are involved in pathogenesis. These include the escape of the mucociliary elevator, evasion of host immunity, survival in environments with scarce nutrients, and finally participation in polymicrobial infections. Lipooligosaccharide branching, proteinous adhesins, metabolic adaption to nutrient availability and many scavenging systems are implicated in these processes. Interestingly, genome-based studies comparing virulent and commensal strains have identified many hypothetical proteins as virulence determinants, suggesting that much regarding the molecular pathogenesis of NTHi remains to be solved. Summary NTHi is an opportunistic pathogen and highly specialized colonizer of the human respiratory tract that has developed intricate mechanisms to establish growth and survival in the human host. Continued research is needed to further elucidate NTHi host–pathogen and pathogen–pathogen interactions. Keywords Haemophilus influenzae, immune evasion, molecular pathogenesis, niche adaptation, polymicrobial infections
INTRODUCTION Haemophilus influenzae is a commensalistic Gramnegative species of the human upper respiratory tract microbiota and a frequent cause of mucosal infections [1]. As a model organism, H. influenzae has been involved in several major scientific achievements throughout history; it was the source of the isolation of the first restriction enzyme [2], it was used to develop the first animal model for bacterial meningitis [3] and was also the first free-living organism to have its genome fully sequenced [4]. Furthermore, the first licensed glycoconjugate vaccine for use in humans was developed against encapsulated H. influenzae type b (Hib) [5], and, more recently, H. influenzaederived protein D has successfully been employed as the carrier protein for a licensed pneumococcal conjugate vaccine [6]. www.co-infectiousdiseases.com
Non-typeable, that is, uncapsulated H. influenzae (NTHi) is a prevalent cause of acute and recurrent otitis media in children and exacerbations in chronic obstructive pulmonary disease (COPD) patients [7–11]. It is also the etiological agent of bacterial pulmonary infections in immunocompromised hosts such as lung cancer patients, and occasionally disseminates to cause invasive disease [11–14]. To establish a successful colonization, Medical Microbiology, Department of Laboratory Medicine Malmo¨, Lund University, Malmo¨, Sweden Correspondence to Dr Kristian Riesbeck, Medical Microbiology, Department of Laboratory Medicine Malmo¨, Lund University, Jan Waldenstro¨ms gata 59, SE-205 02 Malmo¨, Sweden. Tel: +46 40 338494; e-mail: [email protected] Curr Opin Infect Dis 2014, 27:268–274 DOI:10.1097/QCO.0000000000000056 Volume 27 Number 3 June 2014
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Haemophilus influenzae pathogenesis Jalalvand and Riesbeck
KEY POINTS NTHi causes mucosal and invasive opportunistic infections in humans to a high socioeconomic cost. NTHi is associated with a large degree of genetic heterogeneity and interstrain DNA exchange. NTHi is highly adapted to colonize the human respiratory tract and evades swift clearance by the host immune system via virulence factors including lipo-oligosaccharide, proteinous adhesins and binding of complement inhibitors. NTHi has developed intricate systems to metabolically respond to changes in the microenvironment. Recent studies have elucidated some of the molecular mechanisms underlying polymicrobial infections involving NTHi and co-pathogens, and these include viral attenuation of the host immune response.
NTHi has to cope with the structural barrier of the mucociliary epithelium, the innate and acquired immunity, intermicrobial competition, and nutrient poor environments. NTHi is highly adapted to these conditions and usually establishes a transient colonization with a turnover rate of 3 months; the host is overtaken by a new strain as the immune system clears the preceding one [15]. As NTHi currently causes the vast majority of H. influenzae infections, this review is primarily focused on recent advances related to the molecular pathogenesis of NTHi.
GENETIC INTRASPECIES HETEROGENEITY H. influenzae is a genetically diverse species with each strain containing between 1765 and 2355 genes (http://www.ncbi.nlm.nih.gov/genome/genomes/ 165). The core-genome present in all strains consists of 1485 genes, or about 75% of the genomic content of any given isolate [16 ]. The supragenome of H. influenzae, however, has been predicted to contain approximately 4500 unique genes. As many strains are naturally competent, it is plausible that a constant and dynamic genetic exchange occurs via uptake and recombination of intraspecies DNA containing unique H. influenzae uptake sequences [17], with individual strains having access to various parts of the supragenome during concomitant colonization of the same host [18 ,19]. This mechanism would allow the bacteria to access a wide variety of genes while still keeping their individual genomes small, thus giving them a fitness advantage. Specific disease-associated genetic elements, as opposed to asymptomatic colonization factors, have &&
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long remained elusive, although some genes have been found to be more prevalent among virulent strains, as has been reviewed [20]. However, when the complete gene content of 210 geographically and clinically diverse NTHi strains was recently compared, 149 genes were identified to be significantly associated with either virulence or commensalism [16 ]. Interestingly, the 28 genes that were more likely to be found in virulent strains were not any of the well characterized virulence factors involved in adherence, lipo oligosaccharide (LOS) biosynthesis, or immune evasion. Indeed, most were hypothetical proteins indicating that the principal virulence determinants may have eluded researchers thus far. &&
HOST COLONIZATION AND PERSISTENCE The initial step of successful colonization and subsequent infection is adherence to the host tissue. To circumvent mechanical clearance by the mucosal epithelium, NTHi uses four main strategies: perturbance of the mucociliary elevator, adherence to the epithelium, formation of microcolonies and biofilm, and/or invasion of epithelial cells. One mechanism underlying NTHi-mediated decreased ciliary beating and detachment of ciliated cells was recently shown to involve the activation of host protein kinase C epsilon [21]. However, disintegrity of ciliated cells is not always observed, as reported by other investigators using extended time co-culture (1–10 days) with NTHi and primary human respiratory epithelial cells [22]. Instead, bacteria appeared to seek refuge in paracellular foci to evade the mucociliary elevator. Intercellular bacteria were seemingly associated with the basal cell layers, where junctional disorganization was observed. Both these strategies can plausibly be implemented during human colonization. Epithelial cells are connected to the structural scaffold of the underlying extracellular matrix (ECM) via a range of basal surface structures including integrins [23]. Should these interactions be disrupted (as observed in the studies above), or the epithelium be damaged by viral infections or chronic inflammation, ECM proteins become a viable target for adherence by pathogens [24]. Like other respiratory tract pathogens, NTHi has evolved strategies to bind to ECM proteins. We recently reported that NTHi protein F, a homolog of streptococcal laminin-binding proteins Lbp/Lmb, is a novel adhesin that directly mediates bacterial binding to laminin and primary human bronchial epithelial cells [25 ]. These results are in analogy with NTHi adhesins protein E and Hap that have previously been reported to interact with both ECM proteins as
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well as host cells [26–28]. Interestingly, protein F (gene also annotated as hfeA) has been found to be crucial for successful NTHi infection in vivo [29 ]. Animal models with mice, rats, and chinchillas have traditionally been used to mimic human NTHi infections, but the relevance of the data obtained in these studies will always remain an issue because of the human-specific nature of the pathogen. In a new and exciting study, Winokur et al. [30 ] developed a human nasopharyngeal colonization model to study the virulence of NTHi in its natural host. Thirteen selected phase-variable genes were monitored during a 6-day period of human colonization and significant changes were observed in two genes, phosphorylcholine kinase licA and IgA1-protease igaB [31 ]. LicA incorporates exogenously acquired phosphorylcholine into LOS, an alteration that mediates increased adherence and entry into bronchial epithelial cells. The in-vivo selection of subpopulations shifting to phase-on expression of licA suggests that this mechanism is of importance during early colonization. Invasion and persistence of NTHi in epithelial cells have been extensively reviewed [32]. There is also increasing evidence showing that NTHi may reside in lymphoid tissues and act as a reservoir for recurrent infections [33,34]. Additional mechanisms of H. influenzae persistence are reported to involve classic toxin–antitoxin systems that may arrest cell proliferation during stressful conditions via mRNA targeting and result in dormant persister cells [35]. In a recent report, NTHi IgA1 proteases were implicated in internalization and intracellular persistence in bronchial epithelial cells [36]. IgA1 protease is a secreted virulence factor with high specificity for the predominant mucosal immunoglobulin, secretory IgA1, which it cleaves in the hinge region to circumvent the host humoral response. As a testament to its importance for pathogenesis, the protease was one of two phase-variable genes shown to be significantly upregulated during experimental human colonization [31 ]. Two variants of IgA1 protease have been identified in NTHi, IgaA and, more recently, IgaB [36]. Authors found that IgaA was required for optimal invasion of epithelial cells, whereas IgaB was needed for optimal intracellular persistence. Intriguingly, it is unclear how IgaA enhances NTHi internalization. Possible mechanisms could be a yet unidentified enzymatic function that the secreted protease exerts on bacterial and/or host cells, or interaction between the outer membrane-integrated residual IgaA translocator domain and epithelial cells. Albeit previously controversial, the formation of NTHi biofilm in vivo is nowadays generally acknowledged by most researchers. NTHi residing in &&
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biofilms, as compared with planktonic subsistence, acquire a series of advantages including enhanced resistance to antimicrobial compounds and host immune effectors [37,38 ]. The current understanding of NTHi biofilms has recently been excellently reviewed in several publications [37,38 ,39,40]. &&
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NICHE ADAPTABILITY AND NUTRIENT ACQUISITION The human nasopharynx is a nutrient-poor environment for bacteria, especially during inflammation when the host sequesters essential nutrients [41 ]. To establish a successful colonization of the respiratory tract, otopathogens rely on their ability to adapt to this niche, scavenge scarce nutrients, and respond metabolically to changes in the environment. A recent study found that H. influenzae carbonic anhydrase is essential for growth in low CO2 concentrations and important for prolonged intracellular survival in phagosomes, suggesting a role for this enzyme in bacterial adaption to different CO2 conditions and acidic pH [42]. The pathogen also utilizes urease, found to be more prevalent in disease-causing isolates than commensals, to survive in acid environments [43,44]. Other systems involved in sensing the microenvironment within the host include the multifunctional Sap-transporter, possibly via detection of host molecules as well as heme accessibility [45,46]. As alluded to by its name, Haemophilus lacks the ability to synthesize heme that is essential for its survival. Heme is thus acquired exogenously and the pathogen has developed many ways for sensing extracellular heme (and iron) and altering its gene expression to correctly respond to the availability of the metabolite [47,48]. Expectedly, disruption of these systems results in decreased virulence [46–48]. One of the central transcriptional regulators involved in iron-acquisition is the ferric uptake regulator (Fur) protein. The Fur regulon of NTHi was recently defined and shown to contain 73 genes [49]. Interestingly, authors found that in addition to iron uptake and utilization genes, IgA1 protease was also regulated by Fur, suggesting that the bacterium has coupled low iron concentrations to the mucosal milieu. An isogenic Dfur mutant was found to be attenuated in vivo. Fur is not the only regulatory responder to low iron concentrations. Recently, the core modulon (identical in five different isolates) of H. influenzae adaptation to low levels of iron/heme was defined in vitro and verified in vivo [50 ]. In addition to the 55 genes that were similarly regulated in all strains, authors identified approximately 200 non-core &
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Haemophilus influenzae pathogenesis Jalalvand and Riesbeck
genes that they hypothesize are important for environmental adaptation. The conditions in the lower respiratory tract of COPD patients differ significantly from those in the nasopharynx, with mucus hypersecretion (that probably causes hypoxic microenvironments), failure of cilia-mediated clearance, high cytokine levels, excessive phagocyte presence, epithelial disintegrity, and oxidant/antioxidant imbalances [51]. The genetic adaptation of NTHi populations to these changes have been examined and 15 genetic islands have been identified to be more prevalent in COPD isolates as compared with commensal nasopharyngeal strains, including genes encoding the IgA1 protease, high-molecular-weight adhesins, and urease [52]. Interestingly, some of these genes had previously been observed to be transcriptionally upregulated during bacterial growth in sputum obtained from COPD patients, suggesting in-situ selection for both genetically and transcriptionally adapted strains [53]. Other factors that potentially contribute to the etiology of exacerbations in COPD patients include bacterial oxidative stress responses and impaired host defenses targeting NTHi [53,54]. The topic of reactive oxygen species-resisting NTHi mechanisms has been thoroughly reviewed recently [55 ]. &
INFLAMMATION AND IMMUNE EVASION In recent years, great achievements have been made in the molecular understanding of NTHi-mediated inflammation processes during otitis media and COPD exacerbations [56,57]. The pathogenesis differs in these conditions, with the balance being shifted toward immune suppression and immune activation in otitis media and COPD, respectively. Nevertheless, evading the host innate immunity, particularly the complement system, is essential for all human bacterial pathogens [58]. NTHi relies on two main strategies for complement evasion: camouflage using branched LOS that covers the bacterial surface or binding of host complement regulator proteins. By attaching a variety of compounds to the LOS core, including scavenged host-specific sialic acid and phosphorylcholine, NTHi blocks the accessibility of bactericidal antibodies to its other surface structures, thus inhibiting complement activation via the classical pathway [59,60 ,61 ,62,63]. Interestingly, this mechanism has shown to be induced in vivo and during exposure to human serum [31 ,60 ,61 ,62]. Other host– pathogen interactions that LOS has been implicated in include biofilm formation and resistance to antimicrobial peptides [64 ]. We have previously shown that acquisition of the host complement inhibitor vitronectin is &
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important for NTHi serum resistance. By using a proteomic approach to identify vitronectin-binding NTHi proteins, we found that Haemophilus protein F mediates complement evasion via sequestration of vitronectin at the bacterial surface [65 ]. &
POLYMICROBIAL INFECTIONS Recent insight into bacterial cell–cell interactions has shown stunning displays of specific and speciesunique secretory responses to direct stimuli [66 ]. It is therefore not surprising that mixed pneumococcal/NTHi otitis media are suggested to be clinically distinct infections compared with single organism otitis media [67]. Co-colonization/infections have long been observed with the otopathogens NTHi, pneumococci, and Moraxella catarrhalis [68–74], but some studies have also reported antagonistic relationships between them [75,76]. Moreover, secondary NTHi infections are commonly detected in patients infected by viral pathogens such as influenza A virus (IAV), rhinovirus, and human respiratory syncytial virus [71,77–80]. It could be that NTHi is particularly well adapted to interplay with other bacteria as well as to host microenvironments affected by primary viral infections. The understanding of the molecular mechanisms underlying polymicrobial superinfections has improved during the last years. One such mechanism was recently unfolded as investigators showed that rhinovirus-induced degradation of the host signaling pathway adaptor protein IRAK-1 attenuates the TLR2-mediated response to NTHi [81 ]. This results in delayed neutrophil recruitment that hinders swift bacterial clearance, providing one explanation as to why rhinoviral/NTHi superinfections are frequently seen clinically. We recently shed light on the physiological interplay between NTHi and group A streptococci (GAS), two pathogens whose presence has been shown to correlate in children suffering from acute pharyngotonsillitis [82,83 ]. We showed that NTHiderived outer membrane vesicles (OMVs) carried functional b-lactamase that protected GAS from amoxicillin-mediated killing. Interestingly, NTHi OMV shedding is seen in several infection models, suggesting that they may play important roles during pathogenesis [22,45]. In a seminal article published by Wong et al. [29 ], investigators employed transposon insertion sequencing to define the NTHi genes needed during single organism infection, co-infection with IAV, and the core set of genes required during both conditions. In addition to observing that IAV made mice highly susceptible to NTHi infections, authors noted that several (but not all) bacterial genes
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involved in defense against various stress conditions were dispensable in co-infections, suggesting that IAV (like rhinovirus) attenuates the host immune response to NTHi. Other interesting findings included the bacterial adaptation to the changes in nutrient access brought on by the viral infection, including alterations in stress responses. These data provide valuable insight in to the idiosyncrasies of polymicrobial infections, and the continued elucidation of the dynamic molecular interactions augmenting NTHi disease in polymicrobial settings will be highly interesting to follow.
Conflicts of interest There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
CONCLUSION NTHi is a remarkable pathogen in many aspects. In contrast to many of its co-pathogens, NTHi does not have any known cytotoxic effectors or exotoxins, secreted proteolytic enzymes (aside from the IgA1specific endopeptidases), lipases, super antigens, injectisome secretion systems, or a polysaccharide capsule. Still it manages to efficiently outcompete other microbes and colonize the upper respiratory tract of children and the lower respiratory tract of immunocompromised adults. How does it do it? NTHi has developed advanced and efficient scavenging systems for obtaining nutrients in the human airways. Indeed, it has been reported that the pathogen produces siderophore-utilization proteins without producing siderophores [84], meaning it steals iron from other bacterial species that in turn steal it from the host! Furthermore, NTHi scavenges essential and host-specific nutrients, effectively saving the cost of synthesizing these products. The price of the loss of biosynthesis enzymes is niche dependency, as NTHi can only sustain prolonged growth in the human respiratory tract. We have gained increasing insight into other virulence mechanisms of NTHi. Investigators have shown that NTHi employs an array of multifunctional proteins, including protein D, protein E, protein F, the Sap-transporter and the heme-binding proteins HbpA and TehB, a display of highly economic use of proteins [25 ,26,27,45,46,65 ,85– 89]. Moreover, genetic heterogeneity and accessibility to a diverse supragenome has provided NTHi with additional fitness benefits as each isolate can maintain a relatively small genome and still be adaptable to environmental selection [16 ]. To summarize, NTHi is a highly adapted human-specific commensal whose molecular pathogenesis is not yet fully understood. The clinical burden of this opportunistic pathogen encourages more research to help combat infections caused by it. &
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Acknowledgements This work was supported by grants from the Alfred O¨sterlund, the Anna and Edwin Berger, Greta and Johan Kock foundations, the Swedish Medical Research Council (grant number 521-2010-4221, www.vr.se), the Physiographical Society (Forssman’s Foundation), and Ska˚ne County Council’s research and development foundation.
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Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. King P. Haemophilus influenzae and the lung (Haemophilus and the lung). Clin Transl Med 2012; 1:10. 2. Smith HO, Wilcox KW. A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J Mol Biol 1970; 51:379–391. 3. Smith AL, Smith DH, Averill DR Jr, et al. Production of Haemophilus influenzae b meningitis in infant rats by intraperitoneal inoculation. Infect Immun 1973; 8:278–290. 4. Fleischmann RD, Adams MD, White O, et al. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 1995; 269:496– 512. 5. Goldblatt D. Conjugate vaccines. Clin Exp Immunol 2000; 119:1–3. 6. Forsgren A, Riesbeck K, Janson H. Protein D of Haemophilus influenzae: a protective nontypeable H. influenzae antigen and a carrier for pneumococcal conjugate vaccines. Clin Infect Dis 2008; 46:726–731. 7. Casey JR, Kaur R, Friedel VC, Pichichero ME. Acute otitis media otopathogens during 2008 to 2010 in Rochester, New York. Pediatr Infect Dis J 2013; 32:805–809. 8. Pichichero ME. Otitis media. Pediatr Clin North Am 2013; 60:391–407. 9. Gkentzi D, Slack MP, Ladhani SN. The burden of nonencapsulated Haemophilus influenzae in children and potential for prevention. Curr Opin Infect Dis 2012; 25:266–272. 10. Dunne EM, Smith-Vaughan HC, Robins-Browne RM, et al. Nasopharyngeal microbial interactions in the era of pneumococcal conjugate vaccination. Vaccine 2013; 31:2333–2342. 11. Agrawal A, Murphy TF. Haemophilus influenzae infections in the H. influenzae type b conjugate vaccine era. J Clin Microbiol 2011; 49:3728–3732. 12. Laroumagne S, Lepage B, Hermant C, et al. Bronchial colonisation in patients with lung cancer: a prospective study. Eur Respir J 2013; 42:220–229. 13. Livorsi DJ, Macneil JR, Cohn AC, et al. Invasive Haemophilus influenzae in the United States, 1999-2008: epidemiology and outcomes. J Infect 2012; 65:496–504. 14. Resman F, Ristovski M, Ahl J, et al. Invasive disease caused by Haemophilus influenzae in Sweden 1997-2009; evidence of increasing incidence and clinical burden of nontype b strains. Clin Microbiol Infect 2011; 17:1638– 1645. 15. Kaur R, Chang A, Xu Q, et al. Phylogenetic relatedness and diversity of nontypable Haemophilus influenzae in the nasopharynx and middle ear fluid of children with acute otitis media. J Med Microbiol 2011; 60:1841–1848. 16. Eutsey RA, Hiller NL, Earl JP, et al. Design and validation of a supragenome && array for determination of the genomic content of Haemophilus influenzae isolates. BMC Genomics 2013; 14:484. This article identifies a wide array of genetic elements uniquely associated with virulence or commensalism. 17. Mell JC, Hall IM, Redfield RJ. Defining the DNA uptake specificity of naturally competent Haemophilus influenzae cells. Nucleic Acids Res 2012; 40:8536–8549. 18. Power PM, Bentley SD, Parkhill J, et al. Investigations into genome diversity of & Haemophilus influenzae using whole genome sequencing of clinical isolates and laboratory transformants. BMC Microbiol 2012; 12:273. This study provides a conclusive phylogenetic tree of H. influenzae strains and shows that capsulated isolates are largely grouped in two clonal lineages. 19. Mell JC, Shumilina S, Hall IM, Redfield RJ. Transformation of natural genetic variation into Haemophilus influenzae genomes. PLoS Pathog 2011; 7:e1002151. 20. Garmendia J, Marti-Lliteras P, Moleres J, et al. Genotypic and phenotypic diversity of the noncapsulated Haemophilus influenzae: adaptation and pathogenesis in the human airways. Int Microbiol 2012; 15:159–172.
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Haemophilus influenzae pathogenesis Jalalvand and Riesbeck 21. Bailey KL, LeVan TD, Yanov DA, et al. Nontypeable Haemophilus influenzae decreases cilia beating via protein kinase C epsilon. Respir Res 2012; 13:49. 22. Ren D, Nelson KL, Uchakin PN, et al. Characterization of extended co-culture of nontypeable Haemophilus influenzae with primary human respiratory tissues. Exp Biol Med (Maywood) 2012; 237:540–547. 23. Singh B, Fleury C, Jalalvand F, Riesbeck K. Human pathogens utilize host extracellular matrix proteins laminin and collagen for adhesion and invasion of the host. FEMS Microbiol Rev 2012; 36:1122–1180. 24. Millares L, Marin A, Garcia-Aymerich J, et al. Specific IgA and metalloproteinase activity in bronchial secretions from stable chronic obstructive pulmonary disease patients colonized by Haemophilus influenzae. Respir Res 2012; 13:113. 25. Jalalvand F, Su YC, Morgelin M, et al. Haemophilus influenzae protein F & mediates binding to laminin and human pulmonary epithelial cells. J Infect Dis 2013; 207:803–813. Identification of a novel adhesin that promotes bacterial binding to epithelial cells and the ECM. 26. Singh B, Al-Jubair T, Morgelin M, et al. The unique structure of Haemophilus influenzae protein E reveals multiple binding sites for host factors. Infect Immun 2013; 81:801–814. 27. Hallstrom T, Singh B, Resman F, et al. Haemophilus influenzae protein E binds to the extracellular matrix by concurrently interacting with laminin and vitronectin. J Infect Dis 2011; 204:1065–1074. 28. Meng G, Spahich N, Kenjale R, et al. Crystal structure of the Haemophilus influenzae Hap adhesin reveals an intercellular oligomerization mechanism for bacterial aggregation. Embo J 2011; 30:3864–3874. 29. Wong SM, Bernui M, Shen H, Akerley BJ. Genome-wide fitness profiling && reveals adaptations required by Haemophilus in coinfection with influenza A virus in the murine lung. Proc Natl Acad Sci U S A 2013; 110:15413–15418. This article identifies the essential NTHi genes required for single and co-infection with influenza A virus in a murine model. 30. Winokur PL, Chaloner K, Doern GV, et al. Safety and immunological out&& comes following human inoculation with nontypeable Haemophilus influenzae. J Infect Dis 2013; 208:728–738. This article shows developement of a human colonization model for NTHi. 31. Poole J, Foster E, Chaloner K, et al. Analysis of nontypeable Haemophilus && influenzae phase-variable genes during experimental human nasopharyngeal colonization. J Infect Dis 2013; 208:720–727. The results from experimental human coloninzation by NTHi show the relative importance of a selected group of phase-variable genes. 32. Clementi CF, Murphy TF. Nontypeable Haemophilus influenzae invasion and persistence in the human respiratory tract. Front Cell Infect Microbiol 2011; 1:1. 33. Stepinska M, Olszewska-Sosinska O, Lau-Dworak M, et al. Identification of intracellular bacteria in adenoid and tonsil tissue specimens: the efficiency of culture versus fluorescent in situ hybridization (FISH). Curr Microbiol 2014; 68:21–29. 34. Singh K, Nordstrom T, Morgelin M, et al. Haemophilus influenzae resides in tonsils and uses immunoglobulin D binding as an evasion strategy. J Infect Dis 2013. [Epub ahead of print] 35. Ren D, Walker AN, Daines DA. Toxin-antitoxin loci vapBC-1 and vapXD contribute to survival and virulence in nontypeable Haemophilus influenzae. BMC Microbiol 2012; 12:263. 36. Clementi CF, Hakansson AP, Murphy TF. Internalization and trafficking of nontypeable Haemophilus influenzae in human respiratory epithelial cells and roles of IgA1 proteases for optimal invasion and persistence. Infect Immun 2014; 82:433–444. 37. Bakaletz LO. Bacterial biofilms in the upper airway: evidence for role in pathology and implications for treatment of otitis media. Paediatr Respir Rev 2012; 13:154–159. 38. Langereis JD, Hermans PW. Novel concepts in nontypeable Haemophilus && influenzae biofilm formation. FEMS Microbiol Lett 2013; 346:81–89. This is the review summing up the current understanding of NTHi biofilm formation. 39. Swords WE. Quorum signaling and sensing by nontypeable Haemophilus influenzae. Front Cell Infect Microbiol 2012; 2:100. 40. Swords WE. Nontypeable Haemophilus influenzae biofilms: role in chronic airway infections. Front Cell Infect Microbiol 2012; 2:97. 41. Krismer B, Liebeke M, Janek D, et al. Nutrient limitation governs Staphylo& coccus aureus metabolism and niche adaptation in the human nose. PLoS Pathog 2014; 10:e1003862. This article shows the identification of nutrient availability in the nasopharynx and subsequent development of a synthetic nasal medium supporting bacterial growth. 42. Langereis JD, Zomer A, Stunnenberg HG, et al. Nontypeable Haemophilus influenzae carbonic anhydrase is important for environmental and intracellular survival. J Bacteriol 2013; 195:2737–2746. 43. Zhang L, Patel M, Xie J, et al. Urease operon and urease activity in commensal and disease-causing nontypeable Haemophilus influenzae. J Clin Microbiol 2013; 51:653–655. 44. Murphy TF, Brauer AL. Expression of urease by Haemophilus influenzae during human respiratory tract infection and role in survival in an acid environment. BMC Microbiol 2011; 11:183. 45. Raffel FK, Szelestey BR, Beatty WL, Mason KM. The Haemophilus influenzae Sap transporter mediates bacterium-epithelial cell homeostasis. Infect Immun 2013; 81:43–54.
46. Vogel AR, Szelestey BR, Raffel FK, et al. SapF-mediated heme-iron utilization enhances persistence and coordinates biofilm architecture of Haemophilus. Front Cell Infect Microbiol 2012; 2:42. 47. Steele KH, O’Connor LH, Burpo N, et al. Characterization of a ferrous ironresponsive two-component system in nontypeable Haemophilus influenzae. J Bacteriol 2012; 194:6162–6173. 48. Hempel RJ, Morton DJ, Seale TW, et al. The role of the RNA chaperone Hfq in Haemophilus influenzae pathogenesis. BMC Microbiol 2013; 13:134. 49. Harrison A, Santana EA, Szelestey BR, et al. Ferric uptake regulator and its role in the pathogenesis of nontypeable Haemophilus influenzae. Infect Immun 2013; 81:1221–1233. 50. Whitby PW, Vanwagoner TM, Seale TW, et al. Comparison of transcription of & the Haemophilus influenzae iron/heme modulon genes in vitro and in vivo in the chinchilla middle ear. BMC Genomics 2013; 14:925. This is an interesting insight into the iron/heme-limitation modulon of NTHi. 51. Doring G, Parameswaran IG, Murphy TF. Differential adaptation of microbial pathogens to airways of patients with cystic fibrosis and chronic obstructive pulmonary disease. FEMS Microbiol Rev 2011; 35:124– 146. 52. Zhang L, Xie J, Patel M, et al. Nontypeable Haemophilus influenzae genetic islands associated with chronic pulmonary infection. PLoS One 2012; 7:e44730. 53. Qu J, Lesse AJ, Brauer AL, et al. Proteomic expression profiling of Haemophilus influenzae grown in pooled human sputum from adults with chronic obstructive pulmonary disease reveal antioxidant and stress responses. BMC Microbiol 2010; 10:162. 54. Berenson CS, Kruzel RL, Eberhardt E, Sethi S. Phagocytic dysfunction of human alveolar macrophages and severity of chronic obstructive pulmonary disease. J Infect Dis 2013; 208:2036–2045. 55. Harrison A, Bakaletz LO, Munson RS Jr. Haemophilus influenzae and oxida& tive stress. Front Cell Infect Microbiol 2012; 2:40. This is an extensive review of how NTHi copes with exogenous and endogenous oxidative stress. 56. Liu K, Chen L, Kaur R, Pichichero ME. Transcriptome signature in young children with acute otitis media due to nontypeable Haemophilus influenzae. Int Immunol 2013; 25:353–361. 57. Rotta Detto Loria J, Rohmann K, Droemann D, et al. Haemophilus influenzae infection upregulates the NLRP3 inflammasome and leads to caspase-1dependent secretion of interleukin-1beta: a possible pathway of exacerbations in COPD. PLoS One 2013; 8:e66818. 58. Zipfel PF, Hallstrom T, Riesbeck K. Human complement control and complement evasion by pathogenic microbes – tipping the balance. Mol Immunol 2013; 56:152–160. 59. Clark SE, Snow J, Li J, et al. Phosphorylcholine allows for evasion of bactericidal antibody by Haemophilus influenzae. PLoS Pathog 2012; 8:e1002521. 60. Clark SE, Eichelberger KR, Weiser JN. Evasion of killing by human antibody & and complement through multiple variations in the surface oligosaccharide of Haemophilus influenzae. Mol Microbiol 2013; 88:603–618. This article describes the elucidation of the molecular mechanisms behind LOSmediated complement evasion. 61. Langereis JD, Stol K, Schweda EK, et al. Modified lipooligosaccharide & structure protects nontypeable Haemophilus influenzae from IgM-mediated complement killing in experimental otitis media. MBio 2012; 3:e00079-12. This is another article that reveals the molecular mechanisms behind LOSmediated complement evasion. 62. Nakamura S, Shchepetov M, Dalia AB, et al. Molecular basis of increased serum resistance among pulmonary isolates of nontypeable Haemophilus influenzae. PLoS Pathog 2011; 7:e1001247. 63. Apicella MA. Nontypeable Haemophilus influenzae: the role of N-acetyl-5neuraminic acid in biology. Front Cell Infect Microbiol 2012; 2:19. 64. Morey P, Viadas C, Euba B, et al. Relative contributions of lipooligosaccharide && inner and outer core modifications to nontypeable Haemophilus influenzae pathogenesis. Infect Immun 2013; 81:4100–4111. This is a systematic evaluation of the role of various components of LOS in NTHi pathogenesis. 65. Su YC, Jalalvand F, Morgelin M, et al. Haemophilus influenzae acquires & vitronectin via the ubiquitous Protein F to subvert host innate immunity. Mol Microbiol 2013; 87:1245–1266. This article describes the elucidation of the molecular mechanisms behind protein F-mediated complement evasion. 66. Traxler MF, Watrous JD, Alexandrov T, et al. Interspecies interactions stimulate && diversification of the Streptomyces coelicolor secreted metabolome. MBio 2013; 4:e00459-13. This article provides an interesting insight into the bacterial species-specific metabolic change upon cell–cell interaction. 67. Dagan R, Leibovitz E, Greenberg D, et al. Mixed pneumococcal-nontypeable Haemophilus influenzae otitis media is a distinct clinical entity with unique epidemiologic characteristics and pneumococcal serotype distribution. J Infect Dis 2013; 208:1152–1160. 68. Shiri T, Nunes MC, Adrian PV, et al. Interrelationship of Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus colonization within and between pneumococcal-vaccine naive mother-child dyads. BMC Infect Dis 2013; 13:483.
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Pathogenesis and immune response 69. Hong W, Khampang P, Erbe C, et al. Nontypeable Haemophilus influenzae inhibits autolysis and fratricide of Streptococcus pneumoniae in vitro. Microbes Infect 2013. [Epub ahead of print] 70. Ruohola A, Pettigrew MM, Lindholm L, et al. Bacterial and viral interactions within the nasopharynx contribute to the risk of acute otitis media. J Infect 2013; 66:247–254. 71. van den Bergh MR, Biesbroek G, Rossen JW, et al. Associations between pathogens in the upper respiratory tract of young children: interplay between viruses and bacteria. PLoS One 2012; 7:e47711. 72. Tikhomirova A, Kidd SP. Haemophilus influenzae and Streptococcus pneumoniae: living together in a biofilm. Pathog Dis 2013; 69:114–126. 73. Brockson ME, Novotny LA, Jurcisek JA, et al. Respiratory syncytial virus promotes Moraxella catarrhalis-induced ascending experimental otitis media. PLoS One 2012; 7:e40088. 74. Verhaegh SJ, Snippe ML, Levy F, et al. Colonization of healthy children by Moraxella catarrhalis is characterized by genotype heterogeneity, virulence gene diversity and co-colonization with Haemophilus influenzae. Microbiology 2011; 157:169–178. 75. Xu Q, Almudervar A, Casey JR, Pichichero ME. Nasopharyngeal bacterial interactions in children. Emerg Infect Dis 2012; 18:1738–1745. 76. Dahlblom V, Soderstrom M. Bacterial interactions in the nasopharynx – effects of host factors in children attending day-care centers. J Infect Public Health 2012; 5:133–139. 77. Molyneaux PL, Mallia P, Cox MJ, et al. Outgrowth of the bacterial airway microbiome after rhinovirus exacerbation of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2013; 188:1224–1231. 78. Blyth CC, Webb SA, Kok J, et al. The impact of bacterial and viral co-infection in severe influenza. Influenza Other Respir Viruses 2013; 7:168–176. 79. Hishiki H, Ishiwada N, Fukasawa C, et al. Incidence of bacterial coinfection with respiratory syncytial virus bronchopulmonary infection in pediatric inpatients. J Infect Chemother 2011; 17:87–90. 80. Arrevillaga G, Gaona J, Sanchez C, et al. Respiratory syncytial virus persistence in macrophages downregulates intercellular adhesion molecule-1 expression and reduces adhesion of nontypeable Haemophilus influenzae. Intervirology 2012; 55:442–450.
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81. Unger BL, Faris AN, Ganesan S, et al. Rhinovirus attenuates nontypeable Haemophilus influenzae-stimulated IL-8 responses via TLR2-dependent degradation of IRAK-1. PLoS Pathog 2012; 8:e1002969. This is an elucidation of the molecular mechanisms in rhinoviral attenuation of the host response to NTHi. 82. Brook I, Gober AE. Increased recovery of Moraxella catarrhalis and Haemophilus influenzae in association with group A beta-haemolytic streptococci in healthy children and those with pharyngo-tonsillitis. J Med Microbiol 2006; 55:989–992. 83. Schaar V, Uddback I, Nordstrom T, Riesbeck K. Group A streptococci are & protected from amoxicillin-mediated killing by vesicles containing beta-lactamase derived from Haemophilus influenzae. J Antimicrob Chemother 2014; 69:117–120. This article shows bacterial polymicrobial collaboration related to antibiotic pressure. 84. Morton DJ, Turman EJ, Hensley PD, et al. Identification of a siderophore utilization locus in nontypeable Haemophilus influenzae. BMC Microbiol 2010; 10:113. 85. Johnson RW, McGillivary G, Denoel P, et al. Abrogation of nontypeable Haemophilus influenzae protein D function reduces phosphorylcholine decoration, adherence to airway epithelial cells, and fitness in a chinchilla model of otitis media. Vaccine 2011; 29:1211–1221. 86. Mason KM, Raffel FK, Ray WC, Bakaletz LO. Heme utilization by nontypeable Haemophilus influenzae is essential and dependent on Sap transporter function. J Bacteriol 2011; 193:2527–2535. 87. Shelton CL, Raffel FK, Beatty WL, et al. Sap transporter mediated import and subsequent degradation of antimicrobial peptides in Haemophilus. PLoS Pathog 2011; 7:e1002360. 88. Vergauwen B, Elegheert J, Dansercoer A, et al. Glutathione import in Haemophilus influenzae Rd is primed by the periplasmic hemebinding protein HbpA. Proc Natl Acad Sci U S A 2010; 107:13270– 13275. 89. Whitby PW, Seale TW, Morton DJ, et al. Characterization of the Haemophilus influenzae tehB gene and its role in virulence. Microbiology 2010; 156: 1188–1200.
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