Arch Microbiol (2015) 197:105–112 DOI 10.1007/s00203-014-1068-x
ORIGINAL PAPER
Heterogeneity of Bordetella bronchiseptica adenylate cyclase (cyaA) RTX domain Eniko˝ Wehmann · Bernadett Khayer · Tibor Magyar
Received: 30 July 2014 / Revised: 21 October 2014 / Accepted: 24 November 2014 / Published online: 5 December 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Bordetella bronchiseptica is a widespread pathogen, with a broad host range, occasionally including humans. Diverse virulence factors (adhesins, toxins) allow its adaptation to its host, but this property of the adenylate cyclase (cyaA) toxin is not well understood. In this study, we analyzed the repeats-in-toxin domain of B. bronchiseptica cyaA with PCR, followed by restriction fragment length analysis. Of ninety-two B. bronchiseptica strains collected from different hosts and geographic regions, 72 (78.3 %) carried cyaA and four RFLP types (A–D) were established using NarI and SalI. However, in 20 strains, cyaA was replaced with a peptide transport protein operon. A phylogenetic tree based on partial nucleotide sequences of cyaA revealed that group 2 contains strains of specifically human origin, whereas subgroup 1a contains all but one of the strains from pigs. The human strains showed many PCR–RFLP and sequence variants, confirming the clonal population structure of B. bronchiseptica. Keywords B. bronchiseptica · Adenylate cyclase (cyaA) · PCR–RFLP · Phylogenetic analysis · Zoonosis
Introduction Bordetella bronchiseptica is a widespread facultative pathogen that infects various livestock and household, wild, and
Communicated by Erko Stackebrandt. E. Wehmann (*) · B. Khayer · T. Magyar Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Hungária krt. 21, 1143 Budapest, Hungary e-mail: [email protected]
laboratory animals. Among other diseases, it is responsible for atrophic rhinitis in pigs, kennel cough in dogs, snuffles in rabbits, and bronchopneumonia in cats and guinea pigs (Mattoo and Cherry 2005), and increasing numbers of human infections have also been reported, predominantly in immunocompromised patients (Goodnow 1980; Wernli et al. 2011). The genus Bordetella encodes several virulence factors that facilitate and define the diseases it causes. Adhesins (pili, pertactin, and filamentous hemagglutinin) facilitate bacterial adhesion to epithelial cells, whereas toxins (dermonecrotic toxin, adenylate cyclase toxin, and tracheal cytotoxin) are responsible for the development of the specific lesions in the diseases caused by this pathogen (Magyar and Lax 2002). Adenylate cyclase toxin (ACT) is produced by the classical Bordetella species (B. pertussis, B. parapertussis, and B. bronchiseptica) (Carbonetti 2010) and is a bifunctional adenylate cyclase/hemolysin belonging to the repeats-intoxin (RTX) exotoxins (Glaser et al. 1988). Multifunctional ACT binds the CD11b/CD18 receptor and is translocated into the cytosol of eukaryotic cells, where it generates supraphysiological cyclic adenosine monophosphate (cAMP) levels, activated by calmodulin (Hewlett and Donato 2007). This increase in intracellular cAMP suppresses the immune status of monocytes, macrophages, and neutrophils, thereby reducing their effector functions (phagocytosis, generation of inflammatory mediators) (Serezani et al. 2008). ACT obstructs chemiluminescence and chemotaxis (Mattoo et al. 2001), and induces morphological changes in erythrocytes (Vojtova et al. 2006b) and apoptosis in murine macrophages (Khelef et al. 1993). The ACT operon includes five genes: cyaA encodes CyaA, cyaC encodes a protein that posttranslationally palmitoylated CyaA to activate the toxin and the cyaB, D, and E genes are responsible for the secretion of the toxin. However, the cyaA gene
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of some B. bronchiseptica strains has a nonsense mutation or the entire cya operon is replaced with a less-well-known peptide transport protein operon (ptp) (Buboltz et al. 2008). CyaA consists of four functional domains: the adenylate cyclase activity domain (residues 1–400), the hydrophobic channel-forming domain (residues 400–700), the calciumbinding glycine/aspartate-rich nonapeptide-repeat domain (residues 900–1,660), and the C-terminal domain carrying a secretion signal (residue 1,660–end) (Cotter and Miller 2001; Vojtova et al. 2006a). The ACT showed differences when B. pertussis and B. bronchiseptica strains were compared (Betsou et al. 1995). Considering its wide host range, variations among B. bronchiseptica strains can also be presumed. In this study, we examined the diversity within the RTX domain of adenylate cyclase toxin in B. bronchiseptica strains of various origins, including isolates from human cases.
Materials and methods Bacterial strains, phenotypic characterization, and species‑specific PCR Ninety-two strains of B. bronchiseptica, isolated from a diverse range of host species (swine, dog, rabbit, horse, cat, guinea pig, turkey, and human) and geographic areas (53 strains from Hungary and 39 strains from the other countries), were analyzed (Table 1). The strains were stored in a 20 % (wt/vol) skim milk suspension (Lab M; Bury, UK) at −70 °C. They were cultured on Columbia agar (Lab M) plates supplemented with sheep blood (5 % vol/vol) under aerobic conditions at 37 °C for 24 h. All strains were subjected to phenotypic tests for conventional identification, including tests for catalase, indole, oxidase, urease, glucose, lactose, sucrose, and the ability to reduce nitrate to nitrite. We screened for hemolytic activity on Columbia agar plates containing horse or sheep blood (5 % vol/vol). Species-specific PCR was performed as described by Hozbor et al. (1999).
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PCR primers Primers cyaF (5′-GATGAYGTCGTGCTTGCCAATG CTT-3′) and cyaR (5′-ATGCGGATCTCCAGGTCGTT-3′) complementary to the cyaA region, producing a 2,151-bp amplification product, and ptpF (5′-ATCCTGGTGCAACTG AGGTTCTG-3′) and ptpR (5′-AGGTTGTGGGTGATG AACAGCAG-3′) complementary to the ptp region, producing a 959-bp amplification product (Buboltz et al. 2008), were used. Primers dntF (5′-GCGGTACTTGGGATAATAGA-3′) and dntR (5′-ATAAAGATGAATCGGCATTG-3′) complementary to the dnt region produced a 224-bp amplification product (Stepniewska and Markowska-Daniel 2010). The primers were synthesized by Genosys Co. (Sigma, St. Louis, MO, USA). PCR The PCRs were performed in a Swift Mini Instrument (Esco; Hatboro, PA, USA). The reactions contained 2.5 μl of 10× DreamTaq buffer, 200 μM dNTPs, 1.2 mM MgCl2, 2 μl of dimethyl sulfoxide, 15 pmol of each appropriate forward and reverse primer, 1 μl of template DNA, and 1 unit of DreamTaq polymerase (Thermo Scientific, Waltham, MA, USA) in a 25 μl reaction volume. The reaction conditions for the dnt PCR have been described by Stepniewska and Markowska-Daniel (2010). The PCR conditions for cyaA were predenaturation at 94 °C for 3 min, 30 cycles of denaturation at 94 °C for 15 s, and annealing/extension at 68 °C for 2.5 min, with a final extension at 72 °C for 5 min. The PCR conditions for ptp were predenaturation at 95 °C for 5 min, 30 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, and extension at 72 °C for 1 min, with a final extension at 72 °C for 8 min. The amplification products were analyzed in 1.5 % (wt/vol) agarose gels (Lonza; Basel, Switzerland) stained with ethidium bromide. The gels were photographed with a Kodak Gel Logic 212 Imaging System (Rochester, NY, USA). RFLP analysis
PCR followed by restriction fragment length polymorphism (PCR–RFLP) analysis DNA template preparation DNA templates were prepared with the boiling method. One colony was taken from a 1-day-old culture and suspended in 50 μl of sterile distilled water and boiled for 20 min in a Biosan thermomixer (Biosan, Riga, Latvia). After boiling, the samples were centrifuged at 14,000×g for 1 min, and 1 μl of the supernatant was used as the DNA template for PCR analysis.
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The amplicons were purified with the PureLink™ Quick Gel Extraction Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. After the amplified products were concentrated by ethanol precipitation, the cyaA fragment was cleaved with NarI and SalI and the ptp fragment with BglI and NarI, according to the instructions of the manufacturer (Thermo Scientific). The restriction fragments were detected on a 2.5 % (wt/vol) ethidium bromide-stained MetaPhor™ Agarose gel (Lonza). The cyaA RFLP types were generated from the combined results of the RFLP analyses.
Arch Microbiol (2015) 197:105–112 Table 1 B. bronchiseptica strains analyzed in this study
107 Strain
Host
Country of origin
Year of isolation
dnt
cyaA type
ptp
5240 5269 5323 5356a 5463 5493 5500a 5505 5594*,a B58 CE KM22a PV6a 4609 5599 Bb-12 Bg1a Boxtel DAN Bb GF8 Bb IM5 MBORD 676 5339 5340b 5347 5348 5362 5460 5462b 5533 5534 5587 5593 5605 5625b 5626 5628 5629 5639 Bö/11 Bb-11 Bb 335b NCTC 452a Bb 286a MBORD 591a MBORD 685 MBORD 750 MBORD 787 MBORD 843b 5008
Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Rabbit
Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary USA Denmark unknown England The Netherlands Denmark unknown England Australia Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary unknown unknown USA unknown USA USA Denmark The Netherlands Switzerland Hungary
1996 2003 2005 2006 2007 2008 2008 2008 2009 1988 1985 1993 1983 unknown 2010 unknown unknown unknown 1999 unknown unknown unknown 2005 2005 2006 2006 2006 2007 2007 2009 2009 2009 2009 2010 2010 2010 2009 2008 2005 2004 unknown unknown 1910s unknown unknown unknown unknown unknown unknown 1988
+ + + + + + + + + + + + − + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + − − + + + + +
A A A A A A A A A A A A B A A A A A A A A A – – – – – – – – – – – – – – – – – – A – A C B A A A – A
− − − − − − − − − − − − − − − − − − − − − − + + + + + + + + + + + + + + + + + + − + − − − − − − + −
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108 Table 1 continued
dnt dermonecrotic toxin gene; cyaA adenylate cyclase toxin gene; ptp protein transport protein operon * urease-negative strain (Khayer et al. 2011) a partial sequence of cyaA was determined; b partial sequence of ptp was determined
Arch Microbiol (2015) 197:105–112 Strain
Host
Country of origin
Year of isolation
dnt
cyaA type
ptp
5024a 5122 5308a 5601 5602 5612 5614 5622 5630 5631 5633 5636 5648 5653a RB 4032 Bb 9.73a Bb LC2 MBORD 704a MBORD 730 5491 5495a 5497 MBORD 669 MBORD 762a NCTC 8750 Bb CVIa Bb CV2 MBORD 628 MBORD 898 M9 M48a MBORD 635 MBORD 970a MBORD 707a MBORD 901a 5390a Bb ALI Bb DANG Bb DELa Bb REMa Bb VALa
Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Guinea pig Guinea pig Guinea pig Guinea pig Guinea pig Guinea pig Horse Horse Horse Horse Cat Cat Cat Cat Turkey Turkey Human Human Human Human Human Human
Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary France unknown USA Denmark Hungary Hungary Hungary USA Ireland USA unknown unknown USA Germany Hungary Hungary USA The Netherlands USA Germany Hungary unknown unknown unknown unknown unknown
1988 1990 2005 2010 2010 2010 2010 2010 2007 2006 2006 2006 2011 2011 1984 unknown unknown unknown unknown 2008 2008 2008 unknown unknown unknown unknown unknown unknown unknown 1994 1994 unknown unknown unknown unknown 2007 unknown unknown unknown unknown unknown
A A A A A A A A A A A A A A A A A A A A A A A D A A A A A A A A A C B B B A A D B
MBORD 675
Human
Germany
unknown
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + − − − + − −
− − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − −
Sequence and phylogenetic analyses Twenty-five cyaA and five ptp PCR products were sequenced on both strands by Macrogen Europe Ltd (Amsterdam, the Netherlands). Because the PCR product from cyaA was too large to be sequenced directly, we also used an internal primer (cyaAIR: 5′-TGGTTTCGGGTTCGTCCATCA-3′) (Genosys
13
−
B
−
Co.) to determine the cyaA nucleic acid sequence. The nucleotide sequences obtained were aligned, edited, and analyzed with the BioEdit version 7.1.3.0 software (Hall 1999). These sequences and those available in the GenBank database were compared with the ClustalW algorithm (Higgins et al. 1994). A phylogenetic tree was constructed using the MEGA version 6.06 software, with the neighbor-joining criterion, and 1,000
Arch Microbiol (2015) 197:105–112
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rounds of bootstrap resampling were performed to assess the confidence of the tree topology (Tamura et al. 2013). Nucleotide sequence accession numbers At least two samples of each cyaA PCR–RFLP type were sequenced and the partial nucleotide sequences (2,007 bp) of 25 strains were submitted to GenBank (accession numbers KF220450–KF220474). The 909-bp ptp fragment sequences from this study were deposited in GenBank under accession numbers KF220475–KF220479. The sequence data for the strains generated in this study were aligned with the cyaA sequences of the reference strains from GenBank: Bb BIE (human) [AJ303058], Bb CAT1 (cat) [AJ303059], Bb GAN (human) [AJ303062], Bb LAPR (rabbit) [AJ303063], Bb LORD (human) [AJ303064], Bb SEI (human) [AJ303065], Bb FR3539 (human) [FM165654], Bb FR2011 (human) [FM165655], Bb RN014 (human) [FM165656], and Bb FR3474 (human) [FM165657] (Chenal-Francisque et al. 2009); MO149 (human) [HE965805], 1289 (monkey) [HE983626], D445 (human) [HE983627], and Bbr77 (human) [HE983628] (Park et al. 2012); and RB50 (rabbit) [NC_002927] (Parkhill et al. 2003).
Fig. 1 Representative results for cyaA and ptp PCRs: 1 KM22 (pig), 2 PV6 (pig), 3 5340 (dog), 4 5625 (dog), 5 MBORD 843 (dog), 6 MBORD 707 (turkey), 7 MBORD 762 (guinea pig), 8 5024 (rabbit), and 9 5390 (human). M GeneRuler DNA Ladder Mix (Thermo Scientific)
Results Phenotypic and genotypic characterization of B. bronchiseptica strains Bordetella bronchiseptica strains formed small, grayish white colonies on blood agar, and their species-specific biochemical characteristics were determined. All but one of the strains were positive on the urease test. The unusual strain (5594 [pig]) has been described previously (Khayer et al. 2011). A further nine strains (9.8 %) were nitrate negative. The strains examined had varying degrees of hemolytic activity, although no hemolytic activity was observed in some canine strains, especially those isolated in Hungary. All strains yielded species-specific PCR products (237 bp), and all but nine contained the dnt gene (Table 1). PCR–RFLP analysis of cyaA and ptp Table 1 shows the results for the detection of cyaA. Of the 92 B. bronchiseptica strains examined, 72 (78.3 %) carried the cyaA gene (Fig. 1), and four RFLP types (A–D) were observed using the NarI and SalI endonucleases (Fig. 2). Of the 72 cyaA-positive strains, seven were isolated from dogs and none of them originated in Hungary. The most common cyaA profile was type A (84.7 %), which occurred in all host species studied, and all but two of the Hungarian strains were of this type. The strains originating from dogs and humans were diverse representing various types of
Fig. 2 Results of the PCR–RFLP analysis of cyaA with NarI and SalI endonucleases. Type A KM22 (pig), type B PV6 (pig), type C MBORD 707 (turkey), type D MBORD 762 (guinea pig), M1 HyperLadder II (Bioline, London, UK), M2 GeneRuler 100 bp DNA Ladder (Thermo Scientific)
cleavage patterns (dog: A, B, and C; human: A, B, and D). Type B was present in 9.7 % of the cyaA-positive strains, but type C and D occurred only sporadically (Table 1). The ptp region was amplified from 20 (21.7 %) of the 92 B. bronchiseptica strains examined (Table 1). These strains corresponded to the nonhemolytic cyaA-negative strains. Eighteen of these 20 strains were isolated in Hungary, one strain was from Switzerland, and one was from an unknown geographic region. The ptp-positive strains produced uniform patterns when the ptp PCR products were analyzed with the BglI and NarI endonucleases. Sequence and phylogenetic analyses of cyaA and ptp fragments The pairwise alignment of the PCR fragments, equivalent to nucleotide positions 2,827–4,833 of the full-length cyaA sequence, revealed 0.0–3.8 % divergence. The
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Arch Microbiol (2015) 197:105–112
consensus D K A V H V/I D K F S S A Group/ subgroup
D H/RE L S M A K R E S A L V S N P A A V T A
.
.
.
.
.
.
.
.
.
.
.
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1594
1531
1481
D N V A
1 1a
.
.
.
.
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V E .
.
.
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H .
.
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E .
.
.
1b
.
.
.
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.
V .
.
.
.
.
.
.
H D Q T L .
E E G A R R M .
S .
.
.
.
.
.
E .
.
.
1c
.
.
.
.
.
V .
.
.
.
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.
H .
.
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.
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E G A R R M .
S .
.
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.
.
.
E .
.
A .
.
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.
.
.
.
.
.
.
.
1d
.
1441
1387
1365
1363
1357
1347
1336
1256
1253
1248
1246
1245
1244
1238
1237
1220
1218
1215
1213
1207
1184
1164
1157
1146
1144
1140
1125
1113
1104
1059
1055
1050
978
954
positions
1035
Table 2 Amino acid polymorphisms between the cyaA-based phylogenetic groups established in this study
I
.
N L .
R .
.
.
.
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.
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2 2a
.
T S .
Q I
.
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R R V
H R .
.
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P R .
.
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N .
.
Q D T I
A T/A .
R A V
2b
.
T S .
Q I
.
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R R V/A H R .
.
.
.
P R .
.
.
.
.
.
N .
Q D T I
A .
R A V
.
Gray region domain involved in the interaction with the CD11b/CD18; framed regions differences between groups/subgroups of B. bronchiseptica strains according to the cyaA-based phylogenetic analysis in hemolysin-type calcium-binding repeats [nonapeptides]
Fig. 3 Phylogenetic tree based on partial cyaA sequences at nucleotide positions 2,827– 4,833. Filled circle PCR–RFLP type A; open triangle PCR– RFLP type B; filled diamond PCR–RFLP type C; open square PCR–RFLP type D; asterisk sequences from GenBank. The scale bar represents 1 % estimated nucleotide divergence
pairwise alignment of the deduced amino acid sequences, equivalent to amino acid residues 943–1,611 of the fulllength CyaA sequence, showed 0.0–3.8 % divergence among the strains analyzed. The amino acid differences were not only located in the nonapeptide repeats (Table 2). A phylogenetic analysis based on the partial cyaA nucleotide sequence generated two distinct groups, and each was separated into subgroups (Fig. 3). Group 1 contained type A and D strains from diverse host species. Subgroup 1a
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contained only type A strains. Subgroup 1b included two type D strains, and some rabbit- and human-origin type A strains clustered in subgroup 1c. The remaining three strains identified as PCR–RFLP type A clustered into group 1d. Group 2 predominantly contained strains with unique nucleotide sequences and was separated into two subgroups (2a and 2b). All but one of the (PV6) type B strains were assigned to subgroup 2a, and subgroup 2b contained type C strains and PV6 (type B from pig). The
Arch Microbiol (2015) 197:105–112
strains of human origin were located on various branches on the phylogenetic tree, but most of them had a unique nucleotide sequence (Fig. 3). A sequence analysis of five amplified ptp products showed that these ptp sequences were identical to that of canine strain 253 (GenBank: CU633843).
Discussion Although a classical phenotypic analysis of 92 B. bronchiseptica confirmed their identity, a variety of hemolytic activities were detected. Numerous factors can affect hemolysis, including pH, the quality of the erythrocytes, and other environmental influences (the length of incubation, temperature, culture medium; Akerley et al. 1992). However, most variation is caused by Bvg (Bordetella virulence gene)-mediated phenotypic modulation (Cotter and Miller 2001). Nonhemolytic or negligibly hemolytic canine B. bronchiseptica strains have been described previously in various parts of the world, but only a few studies have investigated the genetic basis of this phenomenon (González et al. 2006). The cyaA region (2,151 bp) analyzed here encodes the RTX domain, which is responsible for the bacterial hemolytic activity. The cyaA gene was absent from the nonhemolytic strains originating from dogs in Hungary, presumably because the whole cyaA gene had been deleted (Buboltz et al. 2008). In these strains, cyaA is replaced with a gene encoding a putative ABC transporter permease and oligopeptide/dipeptide transporter protein (ptp), which is 100 % identical to the corresponding sequence in canine strain 253 (HE965806) and some human strains (E013 [JGWY01000025], E012 [JGWX01000045], 99-R0433 [JGWN01000092], 00-P-2796 [JGWH01000167], and 00-P-2730 [JGWG01000244]) recorded in GenBank. We demonstrated that the cyaA gene was only absent in B. bronchiseptica strains in dogs, whereas Chenal-Francisque et al. (2009) described cyaA-deficient strains deriving mainly from humans and occasionally from dogs and rabbits. Experiments with B. bronchiseptica strains in which cyaA is deleted have shown that CyaA is necessary to overcome the innate immunity of the host, possibly by interacting with phagocytic cells, such as neutrophils (Harvill et al. 1999). The strains examined in this study were isolated from diseased hosts, so our work suggests that cyaA is not required for the establishment of B. bronchiseptica infection in the host. Further studies are required to determine whether our cyaA-negative strains isolated from dogs are less virulent than cyaA-positive strains. The presence of the dnt gene was also determined in this study. No strain has been found in which both toxin genes (dnt and cyaA) are absent. Only two canine strains (Bb 286 and MBORD 591) lacked dnt, but they retained cyaA,
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similar to the six dnt-negative strains from humans and the one from a pig. Double-mutant/deleted B. bronchiseptica strains should show reduced viability, but this presumption requires verification. Our PCR–RFLP analysis of the B. bronchiseptica strains revealed that most strains belonged to type A (84.7 %), independent of their host species. The phylogenetic groups established based on the partial cyaA nucleotide sequence reflected their host adaptation to some extent: group 2 largely contained strains of human origin, whereas in subgroup 1a, all but one of the strains were from pigs (Fig. 3). The differences between the phylogenetic groups were also observed at the amino acid level. The B. bronchiseptica strains studied varied between amino acid residues 1,184 and 1,256 (Table 2). This region of B. pertussis CyaA, falling within the region encompassed by amino acids 1,166–1,281, is critical for the interaction of the toxin with the host CD11b/CD18 receptor (El-Azami-El-Idrissi et al. 2003). A genomic analysis of the cyaA C-terminal region showed no polymorphisms in the quite strongly host-adapted B. pertussis and B. parapertussis pathogens (Chenal-Francisque et al. 2009). Thus, the variation we observed in this region may arise from the broad host spectrum of B. bronchiseptica. Several case reports of human diseases caused by B. bronchiseptica have been published, but the source of the infection was rarely examined (Wernli et al. 2011). In our study, B. bronchiseptica strains from humans showed high variability on PCR–RFLP analysis. The seven human strains examined produced three distinct patterns (types A, B, and D). Type B was found most frequently, although it occurred only sporadically among the strains from other host species. Most strains from humans clustered on a separate branch (group 2) of the partial cyaA-based phylogenetic tree, although occasional strains from humans were present in group 1. The heterogeneity of B. bronchiseptica strains from humans was previously examined by ChenalFrancisque et al. (2009), who detected 12 allelic types in 30 cyaA-positive strains isolated from human patients. We have described here a novel allelic type from a B. bronchiseptica strain of human origin (strain 5390), which differs from strain Bb SEI at eight nucleotides in the region analyzed (data not shown). The diversity of the strains of human origin further confirms the clonal population structure of B. bronchiseptica. Acknowledgments This work was supported by the Hungarian Scientific Research Fund (OTKA, K83332). We thank Prof. Alistair Lax (Microbiology Department of King’s College, London, UK) and Karen B. Register (National Animal Disease Center, USDA, Ames, IA, USA) for providing strains. Conflict of interest The authors have no conflicts of interest to declare.
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References Akerley BJ, Monack DM, Falkow S, Miller JF (1992) The bvgAS locus negatively controls motility and synthesis of flagella in Bordetella bronchiseptica. J Bacteriol 174:980–990 Betsou F, Seismiro O, Danchin A, Guiso N (1995) Cloning and sequence of Bordetella bronchiseptica adenylate cyclase-hemolysin-encoding gene: comparison with the Bordetella pertussis gene. Gene 162:165–166 Buboltz AM, Nicholson TL, Parette MR, Hester SE, Parkhill J, Harvill ET (2008) Replacement of adenylate cyclase toxin in a lineage of Bordetella bronchiseptica. J Bacteriol 190:5502–5511 Carbonetti NH (2010) Pertussis toxin and adenylate cyclase toxin: key virulence factors of Bordetella pertussis and cell biology tools. Future Microbiol 5:455–469 Chenal-Francisque V, Caro V, Boursaux-Eude C, Guiso N (2009) Genomic analysis of the adenylate cyclase-hemolysin C-terminal region of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Res Microbiol 160:330–336 Cotter PA, Miller JF (2001) Bordetella. In: Groisman EA (ed) Principles of bacterial pathogenesis. Academic Press, San Diego, pp 619–674 El-Azami-El-Idrissi M, Bauche C, Loucka J, Osicka R, Sebo P, Ladant D, Leclerc C (2003) Interaction of Bordetella pertussis adenylate cyclase with CD11b/CD18: role of toxin acylation and identification of the main integrin interaction domain. J Biol Chem 278:38514–38521 Glaser P, Ladant D, Sezer O, Pichot F, Ullmann A, Danchin A (1988) The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia coli. Mol Microbiol 2:19–30 González GM, Rosales ME, Morales GB, Crespo JAM (2006) Isolation and characterisation of Bordetella bronchiseptica strains from canine origin. Vet Mexico 37:313–325 Goodnow RA (1980) Biology of Bordetella bronchiseptica. Microbiol Rev 44:722–738 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acid S 41:95–98 Harvill ET, Cotter PA, Yuk MH, Miller JF (1999) Probing the function of Bordetella bronchiseptica adenylate cyclase toxin by manipulating host immunity. Infect Immun 67:1493–1500 Hewlett EL, Donato GM (2007) Bordetella toxins. In: Locht C (ed) Bordetella molecular microbiology. Horizon Bioscience, Norfolk, UK, pp 97–118 Higgins D, Thompson J, Gibson T, Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence
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Arch Microbiol (2015) 197:105–112 weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680 Hozbor D, Fouque F, Guiso N (1999) Detection of Bordetella bronchiseptica by the polymerase chain reaction. Res Microbiol 150:333–341 Khayer B, Rónai Z, Wehmann E, Magyar T (2011) Detection of urease-negative Bordetella bronchiseptica from the field. Acta Vet Hung 59:289–293 Khelef N, Zychlinsky A, Guiso N (1993) Bordetella pertussis induces apoptosis in macrophages: role of adenylate cyclase-hemolysin. Infect Immun 61:4064–4071 Magyar T, Lax AJ (2002) Atrophic rhinitis. In: Brogden KA, Guthmiller JM (eds) Polymicrobial diseases. ASM Press, Washington, pp 169–197 Mattoo S, Cherry JD (2005) Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin Microbiol Rev 18:326–382 Mattoo S, Foreman-Wykert AK, Cotter PA, Miller JF (2001) Mechanisms of Bordetella pathogenesis. Front Biosci 6:E168–E186 Park J, Zhang Y, Buboltz AM et al (2012) Comparative genomics of the classical Bordetella subspecies: the evolution and exchange of virulence-associated diversity amongst closely related pathogens. BMC Genomics 13:545 Parkhill J, Sebaihia M, Preston A et al (2003) Comparative analysis of the genome sequences of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Nat Genet 35:32–40 Serezani CH, Ballinger MN, Aronoff DM, Peters-Golden M (2008) Cyclic AMP: master regulator of innate immune cell function. Am J Respir Cell Mol Biol 39:127–132 Ste˛pniewska K, Markowska-Daniel I (2010) Evaluation of PCR test for detection of dermonecrotoxin of Bordetella bronchiseptica. Bull Vet I Pulawy 54:495–499 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30:2725–2729 Vojtova J, Kamanova J, Sebo P (2006a) Bordetella adenylate cyclase toxin: a swift saboteur of host defense. Curr Opin Microbiol 9:69–75 Vojtova J, Kofronova O, Sebo P, Beneda O (2006b) Bordetella adenylate cyclase toxin induces a cascade of morphological changes of sheep erythrocytes and localizes into clusters in erythrocyte membranes. Microsc Res Tech 69:119–129 Wernli D, Emonet S, Schrenzel J, Harbarth S (2011) Evaluation of eight cases of confirmed Bordetella bronchiseptica infection and colonization over a 15-year period. Clin Microbiol Infect 17:201–203
ORIGINAL PAPER
Heterogeneity of Bordetella bronchiseptica adenylate cyclase (cyaA) RTX domain Eniko˝ Wehmann · Bernadett Khayer · Tibor Magyar
Received: 30 July 2014 / Revised: 21 October 2014 / Accepted: 24 November 2014 / Published online: 5 December 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Bordetella bronchiseptica is a widespread pathogen, with a broad host range, occasionally including humans. Diverse virulence factors (adhesins, toxins) allow its adaptation to its host, but this property of the adenylate cyclase (cyaA) toxin is not well understood. In this study, we analyzed the repeats-in-toxin domain of B. bronchiseptica cyaA with PCR, followed by restriction fragment length analysis. Of ninety-two B. bronchiseptica strains collected from different hosts and geographic regions, 72 (78.3 %) carried cyaA and four RFLP types (A–D) were established using NarI and SalI. However, in 20 strains, cyaA was replaced with a peptide transport protein operon. A phylogenetic tree based on partial nucleotide sequences of cyaA revealed that group 2 contains strains of specifically human origin, whereas subgroup 1a contains all but one of the strains from pigs. The human strains showed many PCR–RFLP and sequence variants, confirming the clonal population structure of B. bronchiseptica. Keywords B. bronchiseptica · Adenylate cyclase (cyaA) · PCR–RFLP · Phylogenetic analysis · Zoonosis
Introduction Bordetella bronchiseptica is a widespread facultative pathogen that infects various livestock and household, wild, and
Communicated by Erko Stackebrandt. E. Wehmann (*) · B. Khayer · T. Magyar Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Hungária krt. 21, 1143 Budapest, Hungary e-mail: [email protected]
laboratory animals. Among other diseases, it is responsible for atrophic rhinitis in pigs, kennel cough in dogs, snuffles in rabbits, and bronchopneumonia in cats and guinea pigs (Mattoo and Cherry 2005), and increasing numbers of human infections have also been reported, predominantly in immunocompromised patients (Goodnow 1980; Wernli et al. 2011). The genus Bordetella encodes several virulence factors that facilitate and define the diseases it causes. Adhesins (pili, pertactin, and filamentous hemagglutinin) facilitate bacterial adhesion to epithelial cells, whereas toxins (dermonecrotic toxin, adenylate cyclase toxin, and tracheal cytotoxin) are responsible for the development of the specific lesions in the diseases caused by this pathogen (Magyar and Lax 2002). Adenylate cyclase toxin (ACT) is produced by the classical Bordetella species (B. pertussis, B. parapertussis, and B. bronchiseptica) (Carbonetti 2010) and is a bifunctional adenylate cyclase/hemolysin belonging to the repeats-intoxin (RTX) exotoxins (Glaser et al. 1988). Multifunctional ACT binds the CD11b/CD18 receptor and is translocated into the cytosol of eukaryotic cells, where it generates supraphysiological cyclic adenosine monophosphate (cAMP) levels, activated by calmodulin (Hewlett and Donato 2007). This increase in intracellular cAMP suppresses the immune status of monocytes, macrophages, and neutrophils, thereby reducing their effector functions (phagocytosis, generation of inflammatory mediators) (Serezani et al. 2008). ACT obstructs chemiluminescence and chemotaxis (Mattoo et al. 2001), and induces morphological changes in erythrocytes (Vojtova et al. 2006b) and apoptosis in murine macrophages (Khelef et al. 1993). The ACT operon includes five genes: cyaA encodes CyaA, cyaC encodes a protein that posttranslationally palmitoylated CyaA to activate the toxin and the cyaB, D, and E genes are responsible for the secretion of the toxin. However, the cyaA gene
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of some B. bronchiseptica strains has a nonsense mutation or the entire cya operon is replaced with a less-well-known peptide transport protein operon (ptp) (Buboltz et al. 2008). CyaA consists of four functional domains: the adenylate cyclase activity domain (residues 1–400), the hydrophobic channel-forming domain (residues 400–700), the calciumbinding glycine/aspartate-rich nonapeptide-repeat domain (residues 900–1,660), and the C-terminal domain carrying a secretion signal (residue 1,660–end) (Cotter and Miller 2001; Vojtova et al. 2006a). The ACT showed differences when B. pertussis and B. bronchiseptica strains were compared (Betsou et al. 1995). Considering its wide host range, variations among B. bronchiseptica strains can also be presumed. In this study, we examined the diversity within the RTX domain of adenylate cyclase toxin in B. bronchiseptica strains of various origins, including isolates from human cases.
Materials and methods Bacterial strains, phenotypic characterization, and species‑specific PCR Ninety-two strains of B. bronchiseptica, isolated from a diverse range of host species (swine, dog, rabbit, horse, cat, guinea pig, turkey, and human) and geographic areas (53 strains from Hungary and 39 strains from the other countries), were analyzed (Table 1). The strains were stored in a 20 % (wt/vol) skim milk suspension (Lab M; Bury, UK) at −70 °C. They were cultured on Columbia agar (Lab M) plates supplemented with sheep blood (5 % vol/vol) under aerobic conditions at 37 °C for 24 h. All strains were subjected to phenotypic tests for conventional identification, including tests for catalase, indole, oxidase, urease, glucose, lactose, sucrose, and the ability to reduce nitrate to nitrite. We screened for hemolytic activity on Columbia agar plates containing horse or sheep blood (5 % vol/vol). Species-specific PCR was performed as described by Hozbor et al. (1999).
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PCR primers Primers cyaF (5′-GATGAYGTCGTGCTTGCCAATG CTT-3′) and cyaR (5′-ATGCGGATCTCCAGGTCGTT-3′) complementary to the cyaA region, producing a 2,151-bp amplification product, and ptpF (5′-ATCCTGGTGCAACTG AGGTTCTG-3′) and ptpR (5′-AGGTTGTGGGTGATG AACAGCAG-3′) complementary to the ptp region, producing a 959-bp amplification product (Buboltz et al. 2008), were used. Primers dntF (5′-GCGGTACTTGGGATAATAGA-3′) and dntR (5′-ATAAAGATGAATCGGCATTG-3′) complementary to the dnt region produced a 224-bp amplification product (Stepniewska and Markowska-Daniel 2010). The primers were synthesized by Genosys Co. (Sigma, St. Louis, MO, USA). PCR The PCRs were performed in a Swift Mini Instrument (Esco; Hatboro, PA, USA). The reactions contained 2.5 μl of 10× DreamTaq buffer, 200 μM dNTPs, 1.2 mM MgCl2, 2 μl of dimethyl sulfoxide, 15 pmol of each appropriate forward and reverse primer, 1 μl of template DNA, and 1 unit of DreamTaq polymerase (Thermo Scientific, Waltham, MA, USA) in a 25 μl reaction volume. The reaction conditions for the dnt PCR have been described by Stepniewska and Markowska-Daniel (2010). The PCR conditions for cyaA were predenaturation at 94 °C for 3 min, 30 cycles of denaturation at 94 °C for 15 s, and annealing/extension at 68 °C for 2.5 min, with a final extension at 72 °C for 5 min. The PCR conditions for ptp were predenaturation at 95 °C for 5 min, 30 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, and extension at 72 °C for 1 min, with a final extension at 72 °C for 8 min. The amplification products were analyzed in 1.5 % (wt/vol) agarose gels (Lonza; Basel, Switzerland) stained with ethidium bromide. The gels were photographed with a Kodak Gel Logic 212 Imaging System (Rochester, NY, USA). RFLP analysis
PCR followed by restriction fragment length polymorphism (PCR–RFLP) analysis DNA template preparation DNA templates were prepared with the boiling method. One colony was taken from a 1-day-old culture and suspended in 50 μl of sterile distilled water and boiled for 20 min in a Biosan thermomixer (Biosan, Riga, Latvia). After boiling, the samples were centrifuged at 14,000×g for 1 min, and 1 μl of the supernatant was used as the DNA template for PCR analysis.
13
The amplicons were purified with the PureLink™ Quick Gel Extraction Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. After the amplified products were concentrated by ethanol precipitation, the cyaA fragment was cleaved with NarI and SalI and the ptp fragment with BglI and NarI, according to the instructions of the manufacturer (Thermo Scientific). The restriction fragments were detected on a 2.5 % (wt/vol) ethidium bromide-stained MetaPhor™ Agarose gel (Lonza). The cyaA RFLP types were generated from the combined results of the RFLP analyses.
Arch Microbiol (2015) 197:105–112 Table 1 B. bronchiseptica strains analyzed in this study
107 Strain
Host
Country of origin
Year of isolation
dnt
cyaA type
ptp
5240 5269 5323 5356a 5463 5493 5500a 5505 5594*,a B58 CE KM22a PV6a 4609 5599 Bb-12 Bg1a Boxtel DAN Bb GF8 Bb IM5 MBORD 676 5339 5340b 5347 5348 5362 5460 5462b 5533 5534 5587 5593 5605 5625b 5626 5628 5629 5639 Bö/11 Bb-11 Bb 335b NCTC 452a Bb 286a MBORD 591a MBORD 685 MBORD 750 MBORD 787 MBORD 843b 5008
Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Pig Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog Rabbit
Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary USA Denmark unknown England The Netherlands Denmark unknown England Australia Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary unknown unknown USA unknown USA USA Denmark The Netherlands Switzerland Hungary
1996 2003 2005 2006 2007 2008 2008 2008 2009 1988 1985 1993 1983 unknown 2010 unknown unknown unknown 1999 unknown unknown unknown 2005 2005 2006 2006 2006 2007 2007 2009 2009 2009 2009 2010 2010 2010 2009 2008 2005 2004 unknown unknown 1910s unknown unknown unknown unknown unknown unknown 1988
+ + + + + + + + + + + + − + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + − − + + + + +
A A A A A A A A A A A A B A A A A A A A A A – – – – – – – – – – – – – – – – – – A – A C B A A A – A
− − − − − − − − − − − − − − − − − − − − − − + + + + + + + + + + + + + + + + + + − + − − − − − − + −
13
108 Table 1 continued
dnt dermonecrotic toxin gene; cyaA adenylate cyclase toxin gene; ptp protein transport protein operon * urease-negative strain (Khayer et al. 2011) a partial sequence of cyaA was determined; b partial sequence of ptp was determined
Arch Microbiol (2015) 197:105–112 Strain
Host
Country of origin
Year of isolation
dnt
cyaA type
ptp
5024a 5122 5308a 5601 5602 5612 5614 5622 5630 5631 5633 5636 5648 5653a RB 4032 Bb 9.73a Bb LC2 MBORD 704a MBORD 730 5491 5495a 5497 MBORD 669 MBORD 762a NCTC 8750 Bb CVIa Bb CV2 MBORD 628 MBORD 898 M9 M48a MBORD 635 MBORD 970a MBORD 707a MBORD 901a 5390a Bb ALI Bb DANG Bb DELa Bb REMa Bb VALa
Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Guinea pig Guinea pig Guinea pig Guinea pig Guinea pig Guinea pig Horse Horse Horse Horse Cat Cat Cat Cat Turkey Turkey Human Human Human Human Human Human
Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary France unknown USA Denmark Hungary Hungary Hungary USA Ireland USA unknown unknown USA Germany Hungary Hungary USA The Netherlands USA Germany Hungary unknown unknown unknown unknown unknown
1988 1990 2005 2010 2010 2010 2010 2010 2007 2006 2006 2006 2011 2011 1984 unknown unknown unknown unknown 2008 2008 2008 unknown unknown unknown unknown unknown unknown unknown 1994 1994 unknown unknown unknown unknown 2007 unknown unknown unknown unknown unknown
A A A A A A A A A A A A A A A A A A A A A A A D A A A A A A A A A C B B B A A D B
MBORD 675
Human
Germany
unknown
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + − − − + − −
− − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − −
Sequence and phylogenetic analyses Twenty-five cyaA and five ptp PCR products were sequenced on both strands by Macrogen Europe Ltd (Amsterdam, the Netherlands). Because the PCR product from cyaA was too large to be sequenced directly, we also used an internal primer (cyaAIR: 5′-TGGTTTCGGGTTCGTCCATCA-3′) (Genosys
13
−
B
−
Co.) to determine the cyaA nucleic acid sequence. The nucleotide sequences obtained were aligned, edited, and analyzed with the BioEdit version 7.1.3.0 software (Hall 1999). These sequences and those available in the GenBank database were compared with the ClustalW algorithm (Higgins et al. 1994). A phylogenetic tree was constructed using the MEGA version 6.06 software, with the neighbor-joining criterion, and 1,000
Arch Microbiol (2015) 197:105–112
109
rounds of bootstrap resampling were performed to assess the confidence of the tree topology (Tamura et al. 2013). Nucleotide sequence accession numbers At least two samples of each cyaA PCR–RFLP type were sequenced and the partial nucleotide sequences (2,007 bp) of 25 strains were submitted to GenBank (accession numbers KF220450–KF220474). The 909-bp ptp fragment sequences from this study were deposited in GenBank under accession numbers KF220475–KF220479. The sequence data for the strains generated in this study were aligned with the cyaA sequences of the reference strains from GenBank: Bb BIE (human) [AJ303058], Bb CAT1 (cat) [AJ303059], Bb GAN (human) [AJ303062], Bb LAPR (rabbit) [AJ303063], Bb LORD (human) [AJ303064], Bb SEI (human) [AJ303065], Bb FR3539 (human) [FM165654], Bb FR2011 (human) [FM165655], Bb RN014 (human) [FM165656], and Bb FR3474 (human) [FM165657] (Chenal-Francisque et al. 2009); MO149 (human) [HE965805], 1289 (monkey) [HE983626], D445 (human) [HE983627], and Bbr77 (human) [HE983628] (Park et al. 2012); and RB50 (rabbit) [NC_002927] (Parkhill et al. 2003).
Fig. 1 Representative results for cyaA and ptp PCRs: 1 KM22 (pig), 2 PV6 (pig), 3 5340 (dog), 4 5625 (dog), 5 MBORD 843 (dog), 6 MBORD 707 (turkey), 7 MBORD 762 (guinea pig), 8 5024 (rabbit), and 9 5390 (human). M GeneRuler DNA Ladder Mix (Thermo Scientific)
Results Phenotypic and genotypic characterization of B. bronchiseptica strains Bordetella bronchiseptica strains formed small, grayish white colonies on blood agar, and their species-specific biochemical characteristics were determined. All but one of the strains were positive on the urease test. The unusual strain (5594 [pig]) has been described previously (Khayer et al. 2011). A further nine strains (9.8 %) were nitrate negative. The strains examined had varying degrees of hemolytic activity, although no hemolytic activity was observed in some canine strains, especially those isolated in Hungary. All strains yielded species-specific PCR products (237 bp), and all but nine contained the dnt gene (Table 1). PCR–RFLP analysis of cyaA and ptp Table 1 shows the results for the detection of cyaA. Of the 92 B. bronchiseptica strains examined, 72 (78.3 %) carried the cyaA gene (Fig. 1), and four RFLP types (A–D) were observed using the NarI and SalI endonucleases (Fig. 2). Of the 72 cyaA-positive strains, seven were isolated from dogs and none of them originated in Hungary. The most common cyaA profile was type A (84.7 %), which occurred in all host species studied, and all but two of the Hungarian strains were of this type. The strains originating from dogs and humans were diverse representing various types of
Fig. 2 Results of the PCR–RFLP analysis of cyaA with NarI and SalI endonucleases. Type A KM22 (pig), type B PV6 (pig), type C MBORD 707 (turkey), type D MBORD 762 (guinea pig), M1 HyperLadder II (Bioline, London, UK), M2 GeneRuler 100 bp DNA Ladder (Thermo Scientific)
cleavage patterns (dog: A, B, and C; human: A, B, and D). Type B was present in 9.7 % of the cyaA-positive strains, but type C and D occurred only sporadically (Table 1). The ptp region was amplified from 20 (21.7 %) of the 92 B. bronchiseptica strains examined (Table 1). These strains corresponded to the nonhemolytic cyaA-negative strains. Eighteen of these 20 strains were isolated in Hungary, one strain was from Switzerland, and one was from an unknown geographic region. The ptp-positive strains produced uniform patterns when the ptp PCR products were analyzed with the BglI and NarI endonucleases. Sequence and phylogenetic analyses of cyaA and ptp fragments The pairwise alignment of the PCR fragments, equivalent to nucleotide positions 2,827–4,833 of the full-length cyaA sequence, revealed 0.0–3.8 % divergence. The
13
110
Arch Microbiol (2015) 197:105–112
consensus D K A V H V/I D K F S S A Group/ subgroup
D H/RE L S M A K R E S A L V S N P A A V T A
.
.
.
.
.
.
.
.
.
.
.
.
1594
1531
1481
D N V A
1 1a
.
.
.
.
.
V E .
.
.
.
.
.
H .
.
.
.
.
.
.
E .
.
.
1b
.
.
.
.
.
V .
.
.
.
.
.
.
H D Q T L .
E E G A R R M .
S .
.
.
.
.
.
E .
.
.
1c
.
.
.
.
.
V .
.
.
.
.
.
.
H .
.
.
.
.
.
E G A R R M .
S .
.
.
.
.
.
.
.
.
.
E .
.
A .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1d
.
1441
1387
1365
1363
1357
1347
1336
1256
1253
1248
1246
1245
1244
1238
1237
1220
1218
1215
1213
1207
1184
1164
1157
1146
1144
1140
1125
1113
1104
1059
1055
1050
978
954
positions
1035
Table 2 Amino acid polymorphisms between the cyaA-based phylogenetic groups established in this study
I
.
N L .
R .
.
.
.
.
.
.
.
.
.
.
.
2 2a
.
T S .
Q I
.
.
.
R R V
H R .
.
.
.
P R .
.
.
.
.
.
N .
.
Q D T I
A T/A .
R A V
2b
.
T S .
Q I
.
.
.
R R V/A H R .
.
.
.
P R .
.
.
.
.
.
N .
Q D T I
A .
R A V
.
Gray region domain involved in the interaction with the CD11b/CD18; framed regions differences between groups/subgroups of B. bronchiseptica strains according to the cyaA-based phylogenetic analysis in hemolysin-type calcium-binding repeats [nonapeptides]
Fig. 3 Phylogenetic tree based on partial cyaA sequences at nucleotide positions 2,827– 4,833. Filled circle PCR–RFLP type A; open triangle PCR– RFLP type B; filled diamond PCR–RFLP type C; open square PCR–RFLP type D; asterisk sequences from GenBank. The scale bar represents 1 % estimated nucleotide divergence
pairwise alignment of the deduced amino acid sequences, equivalent to amino acid residues 943–1,611 of the fulllength CyaA sequence, showed 0.0–3.8 % divergence among the strains analyzed. The amino acid differences were not only located in the nonapeptide repeats (Table 2). A phylogenetic analysis based on the partial cyaA nucleotide sequence generated two distinct groups, and each was separated into subgroups (Fig. 3). Group 1 contained type A and D strains from diverse host species. Subgroup 1a
13
contained only type A strains. Subgroup 1b included two type D strains, and some rabbit- and human-origin type A strains clustered in subgroup 1c. The remaining three strains identified as PCR–RFLP type A clustered into group 1d. Group 2 predominantly contained strains with unique nucleotide sequences and was separated into two subgroups (2a and 2b). All but one of the (PV6) type B strains were assigned to subgroup 2a, and subgroup 2b contained type C strains and PV6 (type B from pig). The
Arch Microbiol (2015) 197:105–112
strains of human origin were located on various branches on the phylogenetic tree, but most of them had a unique nucleotide sequence (Fig. 3). A sequence analysis of five amplified ptp products showed that these ptp sequences were identical to that of canine strain 253 (GenBank: CU633843).
Discussion Although a classical phenotypic analysis of 92 B. bronchiseptica confirmed their identity, a variety of hemolytic activities were detected. Numerous factors can affect hemolysis, including pH, the quality of the erythrocytes, and other environmental influences (the length of incubation, temperature, culture medium; Akerley et al. 1992). However, most variation is caused by Bvg (Bordetella virulence gene)-mediated phenotypic modulation (Cotter and Miller 2001). Nonhemolytic or negligibly hemolytic canine B. bronchiseptica strains have been described previously in various parts of the world, but only a few studies have investigated the genetic basis of this phenomenon (González et al. 2006). The cyaA region (2,151 bp) analyzed here encodes the RTX domain, which is responsible for the bacterial hemolytic activity. The cyaA gene was absent from the nonhemolytic strains originating from dogs in Hungary, presumably because the whole cyaA gene had been deleted (Buboltz et al. 2008). In these strains, cyaA is replaced with a gene encoding a putative ABC transporter permease and oligopeptide/dipeptide transporter protein (ptp), which is 100 % identical to the corresponding sequence in canine strain 253 (HE965806) and some human strains (E013 [JGWY01000025], E012 [JGWX01000045], 99-R0433 [JGWN01000092], 00-P-2796 [JGWH01000167], and 00-P-2730 [JGWG01000244]) recorded in GenBank. We demonstrated that the cyaA gene was only absent in B. bronchiseptica strains in dogs, whereas Chenal-Francisque et al. (2009) described cyaA-deficient strains deriving mainly from humans and occasionally from dogs and rabbits. Experiments with B. bronchiseptica strains in which cyaA is deleted have shown that CyaA is necessary to overcome the innate immunity of the host, possibly by interacting with phagocytic cells, such as neutrophils (Harvill et al. 1999). The strains examined in this study were isolated from diseased hosts, so our work suggests that cyaA is not required for the establishment of B. bronchiseptica infection in the host. Further studies are required to determine whether our cyaA-negative strains isolated from dogs are less virulent than cyaA-positive strains. The presence of the dnt gene was also determined in this study. No strain has been found in which both toxin genes (dnt and cyaA) are absent. Only two canine strains (Bb 286 and MBORD 591) lacked dnt, but they retained cyaA,
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similar to the six dnt-negative strains from humans and the one from a pig. Double-mutant/deleted B. bronchiseptica strains should show reduced viability, but this presumption requires verification. Our PCR–RFLP analysis of the B. bronchiseptica strains revealed that most strains belonged to type A (84.7 %), independent of their host species. The phylogenetic groups established based on the partial cyaA nucleotide sequence reflected their host adaptation to some extent: group 2 largely contained strains of human origin, whereas in subgroup 1a, all but one of the strains were from pigs (Fig. 3). The differences between the phylogenetic groups were also observed at the amino acid level. The B. bronchiseptica strains studied varied between amino acid residues 1,184 and 1,256 (Table 2). This region of B. pertussis CyaA, falling within the region encompassed by amino acids 1,166–1,281, is critical for the interaction of the toxin with the host CD11b/CD18 receptor (El-Azami-El-Idrissi et al. 2003). A genomic analysis of the cyaA C-terminal region showed no polymorphisms in the quite strongly host-adapted B. pertussis and B. parapertussis pathogens (Chenal-Francisque et al. 2009). Thus, the variation we observed in this region may arise from the broad host spectrum of B. bronchiseptica. Several case reports of human diseases caused by B. bronchiseptica have been published, but the source of the infection was rarely examined (Wernli et al. 2011). In our study, B. bronchiseptica strains from humans showed high variability on PCR–RFLP analysis. The seven human strains examined produced three distinct patterns (types A, B, and D). Type B was found most frequently, although it occurred only sporadically among the strains from other host species. Most strains from humans clustered on a separate branch (group 2) of the partial cyaA-based phylogenetic tree, although occasional strains from humans were present in group 1. The heterogeneity of B. bronchiseptica strains from humans was previously examined by ChenalFrancisque et al. (2009), who detected 12 allelic types in 30 cyaA-positive strains isolated from human patients. We have described here a novel allelic type from a B. bronchiseptica strain of human origin (strain 5390), which differs from strain Bb SEI at eight nucleotides in the region analyzed (data not shown). The diversity of the strains of human origin further confirms the clonal population structure of B. bronchiseptica. Acknowledgments This work was supported by the Hungarian Scientific Research Fund (OTKA, K83332). We thank Prof. Alistair Lax (Microbiology Department of King’s College, London, UK) and Karen B. Register (National Animal Disease Center, USDA, Ames, IA, USA) for providing strains. Conflict of interest The authors have no conflicts of interest to declare.
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