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Developmental and Comparative Immunology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d c i

Recognition, survival and persistence of Staphylococcus aureus in the model host Tenebrio molitor Jack Dorling a,b,c, Caroline Moraes b,d,e, Jens Rolff b,f,* a

Animal & Plant Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK Fachbereich Biologie, Chemie, Pharmazie, Evolutionary Biology, Freie Universität Berlin, Königin-Luise-Straße 1-3, 14195 Berlin, Germany Medical Sciences Doctoral Training Centre, University of Oxford, Oxford, UK d Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil e Centro Universitário Augusto Motta, Rio de Janeiro, Brazil f Berlin-Brandenburg Institute of Advanced Biodiversity Research (BBIB), Berlin, Germany b c

A R T I C L E

I N F O

Article history: Received 4 April 2014 Revised 18 August 2014 Accepted 21 August 2014 Available online Keywords: Persistence Wall teichoic acids Antimicrobial peptides

A B S T R A C T

The degree of specificity of any given immune response to a parasite is governed by the complexity and variation of interactions between host and pathogen derived molecules. Here, we assess the extent to which recognition and immuno-resistance of cell wall mutants of the pathogen Staphylococcus aureus may contribute to establishment and maintenance of persistent infection in the model insect host, Tenebrio molitor. The cell surface of S. aureus is decorated with various molecules, including glycopolymers such as wall teichoic acid (WTA). WTA is covalently bound to peptidoglycan (PGN) and its absence has been associated with increased recognition of PGN by host receptors (PGRPs). WTA is also further modified by other molecules such as D-alanine (D-alanylation). Both the level of WTA expression and its D-alanylation were found to be important in the mediation of the host–parasite interaction in this model system. Specifically, WTA itself was seen to influence immune recognition, while D-alanylation of WTA was found to increase immuno-resistance and was associated with prolonged persistence of S. aureus in T. molitor. These results implicate WTA and its D-alanylation as important factors in the establishment and maintenance of persistent infection, affecting different critical junctions in the immune response; through potential evasion of recognition by PGRPs and resistance to humoral immune effectors during prolonged exposure to the immune system. This highlights a mechanism by which specificity in this host– parasite interaction may arise. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Upon initial infection, the host recognises microbe-associate molecular patterns (MAMPs) such as the cell wall component peptidoglycan (PGN) or lipopolysaccharide (LPS). These molecules are unique to bacteria, theoretically allowing for some degree of specificity in the induction of the immune response. However, rather than evolving direct resistance to stressors, as is a common strategy in environmental microbes, pathogenic bacteria tend to subvert the immune response (Prajsnar et al., 2012) and hence recognition evasion mechanisms are also likely to be of importance for the establishment of infection, and for continued persistence within the host (Prajsnar et al., 2012).

* Corresponding author. Address: Freie Universitaet Berlin, Fachbereich Biologie, Chemie, Pharmazie, Evolutionary Biology, Königin-Luise-Straße 1-3, 14195 Berlin, Germany. Tel.: +44 114 2224777; fax: +44 114 2220002. E-mail address: [email protected] (J. Rolff).

While specific reactions of the immune system of insects have been reported (Armitage et al., 2014; Dong et al., 2006) this often exceeds that predicted on the basis differential stimulation of Toll and Imd signalling by coarsely defined groups of pathogens such as Gram positive and Gram negative bacteria, and a mechanism explaining the findings remains largely elusive. Moreover, phenotypes consistent with specific immune responses can arise at different critical junctions during an infection, such as at the level of recognition or production of immune effectors (Schmid-Hempel and Ebert, 2002). Specificity of an immune response is, however, not only determined by the host’s immune system but also by properties of the pathogen or parasite. Variation in the exposed surface of bacteria, such as the antigenic properties and accessibility of MAMPs, is important for the recognition of pathogens by the host (Atilano et al., 2011) and in the capability of the pathogen to resist clearance by host immune effectors (immuno-resistance) (Kraus and Peschel, 2008). Bacterial cell wall composition is also important in a number of host–parasite interaction processes, from biofilm formation, to adaptation to intracellular environments and evasion of humoral

http://dx.doi.org/10.1016/j.dci.2014.08.010 0145-305X/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Jack Dorling, Caroline Moraes, Jens Rolff, Recognition, survival and persistence of Staphylococcus aureus in the model host Tenebrio molitor, Developmental and Comparative Immunology (2014), doi: 10.1016/j.dci.2014.08.010

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defences such as lysozyme (Bera et al., 2007). As such, pathogens often exhibit specialised immune evasion and immuno-resistance strategies (Peschel and Sahl, 2006) that may contribute to the specificity of the outcome of a particular host–parasite association. In Gram positive bacteria, PGN is seated within a complex of molecules present at the cell surface (Vollmer et al., 2008) including wall teichoic acids, that restrict access to PGN by host peptidoglycan recognition proteins (PGRPs) such as PGRP-SA and GNBP1 (Gram negative binding protein 1) (Atilano et al., 2011; Tabuchi et al., 2010). Wall teichoic acids (WTA) are thought to contribute to immune evasion by Staphylococcus aureus and other Gram positive pathogens. S. aureus strains deficient for the enzyme TagO (ΔtagO), a glycosyltransferase essential in WTA biosynthesis, display reduced WTA expression levels and greater binding of fluorescent-marker-bound PGRP-SA (Atilano et al., 2011). This mutation is also associated with decreased initial bacterial proliferation within the fruit fly, Drosophila melanogaster. WTA expression could therefore conceivably contribute to lower levels of immune recognition in vivo, and may contribute to establishment and maintenance of persistent infection. WTA is also modified by the addition of N-acetylglucosamine (GlcNAc) and D-alanine to its ribitol- and glycerol-phosphate monomeric subunits (Weidenmaier and Peschel, 2008). Using an in vitro cell free system derived from larvae of the yellow flour beetle, Tenebrio molitor, it was shown that D-alanylation of WTA enhanced the effect of reducing PGRP-SA binding to PGN, and subsequent activation of the Toll-cascade (Kurokawa et al., 2011). Furthermore, in D. melanogaster, purified PGN bound to D-alanylated WTA was recognised more poorly by the host than WTA lacking modification by D-alanine (Tabuchi et al., 2010). Recognition of PGN in insects ultimately results in the production of humoral immune effectors (Kounatidis and Ligoxygakis, 2012). In addition to roles in immune evasion, D-alanylation of WTA has also been implicated in resistance to host immune effectors such as antimicrobial peptides (AMPs) (Kristian et al., 2005). WTA is strongly anionic, and thus has a high affinity for many cationic molecules such as antimicrobial peptides (CAMPs). In T. molitor, the production of several AMPs occurs following infection (Johnston et al., 2014). On stimulation of the Toll cascade, as is typical of Gram positive bacteria, Tenecin 1 and Tenecin 2 are produced (Roh et al., 2009), and of these the defensin-like Tenecin 1 is the only known T. molitor AMP active against Gram positive bacteria (Moon et al., 1994). Such D-alanylation mediated resistance to CAMPs may be of particular importance when considered in the context of persistent infection. AMPs are thought to constitute the ‘central pillar’ of the innate immune response (Kounatidis and Ligoxygakis, 2012), and have been canonically deemed essential for front-line bacterial clearance. However, recent studies in T. molitor have suggested that AMPs are instead used in clearance of bacteria refractory to the host’s constitutive defences (Haine et al., 2008a). AMPs may play a role in controlling or suppressing persistent infection, as they are only expressed after the vast majority (99.5%) of bacterial clearance has already taken place (Haine et al., 2008a; Johnston et al., 2014). Most work has so far focused on the role of WTA or D-alanylation in initial establishment of infection, while little consideration has been given to the wider roles of these molecules in maintenance or persistence of infection. To address such questions, we infected T. molitor individuals with a suite of S. aureus ΔtagO mutants varying quantitatively in the biosynthesis levels of WTA and a D-alanylation deficient (ΔdltABCD) S. aureus strain, all from the RN4220 parent background (Atilano et al., 2010). This system is robust to the high inoculate densities required to study persistence, and S. aureus has been shown to be able to persist within T. molitor for at least 28 days. Combined with the quantitative variation in WTA biosynthesis among ΔtagO mutants used here, this provided a unique

opportunity to study the role of WTA and its modification by D-alanylation in the context of persistent infection. Here, using a suite of purposely generated ΔtagO mutants (Atilano et al., 2010) exhibiting quantitatively different levels of WTA biosynthesis, and an independently generated ΔdltABCD operon mutant lacking the ability to D-alanylate WTA, we attempt to elucidate the contributions of recognition by the host and resistance of the bacteria to host immune effectors to bacterial persistence in vivo.

2. Materials and methods 2.1. Culturing of insects and bacterial strains Insect stocks were raised on a mixed wheat germ/rat chow (Harlan Laboratories, UK) diet supplemented with apple (every 2–3 days), and were kept at 30 °C under a 12:12 h light:dark photoperiod. Beetles were collected as pupae, sexed, and housed individually in grid-boxes. Male beetles, 7–14 days post-eclosion were used for experiments. After inoculation with bacteria, beetles were returned to individual housing until sampling. Bacterial strains were all derivatives of S. aureus strain RN4220 expressing varying levels of WTA at the cell surface, or lacking the ability to D-alanylate WTA (Atilano et al., 2010). Strains used in this study, and associated plasmids, are listed in Table 1. Each strain was grown for experiments in tryptic soy broth (TSB) supplemented with the appropriate antibiotics (see Table 1). After 18 h, 750 μL stationary phase culture was mixed with 750 μL glycerol (25% glycerol final concentration) and stored at −80 °C. For propagation of cultures, fresh stock plates were made up every 3 days from the frozen stock and grown at 30 °C for 20–30 h as above. To initiate cultures, a single colony was sampled from stock plates and used to inoculate 10 mL of tryptic soy broth (TSB) containing strain-appropriate antibiotics. Cultures were grown overnight (~18 h) at 30 °C, shaking at 220 rpm and experimental samples adjusted to the required CFU density as detailed in the supplementary material. 2.2. Experimental infection Bacteria were grown as above, standardised for density (see supplement) and centrifuged at 5000 g for 2 minutes. The bacterial pellet was washed with 1 mL phosphate buffered saline (PBS) and the sample centrifuged at 5000 g for a further 2 minutes. The bacterial pellet was then re-suspended in a final 1 mL of PBS. To quantify the inoculates, a ten-fold dilution series to 1:1 × 107 was prepared and the lowest 2 dilutions plated using glass beads. Plates were then incubated for 20–30 h at 30 °C. CFUs were counted post-experiment to check that standardisation of inoculates had produced the expected number of CFUs using an open-source colony counting programme: OpenCFU (Geissmann, 2013). For injections, 5 μL of bacterial suspension or PBS was injected directly into the haemocoel of individual beetles via pulled glass capillary, between the 4th and 5th abdominal sternites. 2.3. Analysis of Toll-mediated AMP expression Bacteria were grown as detailed above. Inoculates were then adjusted to a density of ~1 × 10 7 CFU in each 5 μL inoculate (1 × 109 CFU mL−1, see supplement). This was then diluted 1:10 in series to ~1 × 104 CFU per inoculate, and 5 μL of each of these 4 dilutions injected into test insects (see Table S1 for numbers of infected insects). Beetles were injected with either the parental S. aureus strain (RN4220WT), a strain deficient in D-alanylation (ΔdltABCD) or a reduced WTA mutant (ptagOG152A) (Table 1) to elicit an immune response. The lowest dilution was then diluted 1:10 a further 4 times

Please cite this article in press as: Jack Dorling, Caroline Moraes, Jens Rolff, Recognition, survival and persistence of Staphylococcus aureus in the model host Tenebrio molitor, Developmental and Comparative Immunology (2014), doi: 10.1016/j.dci.2014.08.010

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Table 1 Strains and plasmids used in this study. Generated by Atilano et al. (2010).

Strain RNPBP4YFP

RNΔtagOPBP4YFP pMAD RNΔtagOPBP4YFP ptagO RNΔtagOPBP4YFP ptagOG152A RNΔdltABCDPBP4YFP Plasmid ptagO ptagOG152A pMAD

% WTAa

Resistance markerc

Abbreviation

Description

100

Kan

RN4220WT

0 90 24 100

Kan, Ery Kan, Ery Kan, Ery Kan

pMAD ptagO ptagOG152A ΔdltABCD

Reference strain (parental strain); restriction-deficient derivative of S. aureus NCTC8325-4 capable of stably maintaining shuttle plasmids. YFP fusion to the C-terminus of PBP4b with a kanamycin resistance cassette. RN4220 PBP4YFP tagO null mutant transformed with ptagO vector RN4220 PBP4YFP tagO null mutant transformed with ptagOG152A vector RN4220 PBP4YFP tagO null mutant transformed with pMAD vector RN4220 PBP4YFP dltABCD operon null mutant. This strain cannot D-alanylate WTA

N/A N/A N/A

Ery Ery Ery

N/A N/A N/A

pMAD vector with insert encoding tagO gene and its promoter region pMAD vector with insert encoding tagO protein with mutation G152A Escherichia coli–S. aureus shuttle vector with a thermosensitive origin of replication for Gram-positive bacteria

Note: PBP4::YFP fusions were used to utilise the kanamycin resistance cassette for bacterial identification post infection. a %WTA refers to the estimated density of WTA at the cell surface, as measured by PGRP-SA binding (Atilano et al., 2011). b PBP4 is penicillin binding protein 4, a non-essential PGN synthesis enzyme. c Kan, kanamycin; Ery, erythromycin.

and 50 μL each of the lowest two dilutions plated onto TSA with appropriate antibiotics and spread with ~20 sterile glass beads. 4 h after initial infection, coincident with a peak in AMP mRNA expression (Johnston et al., 2014), beetles were flash frozen at −80 °C. They were then defrosted individually and dissected to remove fat body. Fat body was transferred immediately to 350 μL of TRIreagent stored on ice. Samples were homogenised using 2 sterile stainless steel ball bearings in a tissue lyser (QIAGEN, Tissue lyser II), spun down, and frozen at −80 °C. To isolate RNA, a TRI-reagent/ chloroform extraction was carried out. For T. molitor see Johnston et al (2014). RNA was then quantified and purity assessed (3 technical replicates per sample) in a Nanodrop 2000c (Thermo-Scientific). RNA was then stored at −80 °C. To synthesise cDNA, 250 ng of RNA was incubated with 250 ng of random hexamer primers in a total volume of 8 μL, at 75 °C for 5 minutes to anneal primers and transferred straight to ice. To this, 2.5 μL 5× RT buffer, 1.25 μL dNTPs, 0.25 μL RNasin (Rnase inhibitor) and 0.5 μL reverse transcriptase were added and the samples incubated for 60 minutes at 37 °C, then at 75 °C for 15 minutes to synthesise cDNA. cDNA was diluted in DEPC treated water and stored at −20 °C. Expression of Tenecin 1 (Ten1), an AMP of T. molitor was used as a proxy for Toll-pathway activation. Tenecin 1 was chosen as it is the only known AMP in T. molitor with anti-Gram positive activity (Chae et al., 2012), and is produced on stimulation of the Toll pathway. The ribosomal protein rpl27a (NCBI accession number X99204.1) was selected as an internal control. qPCR was performed on an Applied Biosystems StepOne platform, using KAPA2G Fast ReadyMix (KAPA Biosystems). Each reaction contained 10 μL 2× KAPA2G Fast ReadyMix (KAPA Biosystems), 0.4 μL of both forward and reverse primer, 2.5 μL cDNA and DEPC treated water to a total volume of 20 μL. The thermal profile was as follows: 2 min 30 s at 95 °C; 40 cycles of 3 s at 95 °C and 15 s at 60 °C followed by melt-curve analysis. Each sample was run in triplicate for each primer set and treatment groups were split across plates. All primers exhibited efficiency coefficients (R2) of greater than 0.95 and less than 1.00 (data not shown). Primers used in this study are given in Table S2. 2.4. Haemolymph antimicrobial activity assay This procedure was modified from Haine et al. (2008a). Following injection of a cohort of beetles (n = 36) with the parental S. aureus strain, 2–16 μL of haemolymph was collected 24 hrs later via a puncture wound in each insect between the head and the thorax, mixed

with an equal volume of anticoagulant buffer (pH 4.5, 0.061 M citric acid, 0.025 M EDTA, 0.216 M NaCl, 0.146 M NaOH) and frozen on dry ice. Haemolymph was defrosted, divided into 3 independent pools of approximately equal volume, comprising haemolymph from 12 beetles each. This was centrifuged at 20,000 g for 20 minutes at 4 °C to remove bacteria, haemocytes and other suspended debris. S. aureus were prepared as previously described and adjusted to ~5 × 105 CFU mL−1 in PBS. Dilutions were plated to determine initial CFU and enumerated the next day. 4 μL of haemolymph/ anticoagulant mix was mixed with 96 μL of the S. aureus bacterial suspension and incubated at 30 °C, shaking at 220 rpm for 2 hours. Dilutions of the samples were plated to determine remaining CFU as above. This was repeated 3 times for each bacterial mutant, with exposure to each of the 3 independent haemolymph pools. 3 control assays (PBS in place of haemolymph) were also conducted for each strain. All plates were incubated at 30 °C for 20–30 h and counted as above. 2.5. CAMP susceptibility assays To determine strain-specific resistance against AMPs, in vitro assays were conducted using commercially synthesised T. molitor Tenecin 1 (Peptide Protein Research Ltd, see supplement for sequence) and the commercially available pexiganan (a kind gift from Dr. Michael Zasloff). Cercropin A was included as a control AMP known not to kill Gram positive bacteria. Susceptibility was determined using a modification of a previous protocol (Wiegand et al., 2008). Briefly, 20-fold concentrated solutions (640 μg mL−1) of each of the AMPs were first prepared in sterile distilled water. 10-fold concentrated solutions were then made up by dilution in 0.02% acetic acid containing 0.4% BSA, to prevent precipitation of the peptides in the growth medium. 2-fold dilutions from 320 μg mL −1 to 25 μg mL−1 were then prepared using 0.01% acetic acid containing 0.2% BSA, and 10 μL added to each test plate (n = 10) in duplicate. All plates were then frozen at −20 °C and defrosted individually for MIC testing. For MIC testing and collection of growth data, bacteria were grown as previously described before being adjusted to 5 × 105 CFU mL−1 in non-cation adjusted Mueller–Hinton (ncMH) broth. 90 μL of S. aureus culture was added to each well containing test peptides and a further 100 μL of bacterial suspension was added to an entire column of the plate as a growth control, and 100 μL of ncMH added to a final column as a sterility control. The 96-well plates were then incubated at 30 °C, shaking, for 18 hours

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100

150

200

250

β

α

α

0

Beetles were injected with ~5 × 106 CFU of the parental strain (RN4220WT, n = 34), the D-alanylation mutant (ΔdltABCD, n = 32) a reduced WTA mutant (ptagOG152A, n = 32), or with sterile PBS (n = 32). Following this, beetles were maintained under standard rearing conditions in isolation. The time points assayed were 1 day, 3 days and 10 days. A total of 10–14 beetles per treatment (strain/time-point combination) were used. At each time point, haemolymph was then extracted via perfusion bleed (Haine et al., 2008a). Samples were diluted in PBS and plated as above to determine CFU counts. Plates were then incubated for 20–30 h at 30 °C, and colonies enumerated using OpenCFU. Inoculates were also plated as before to enumerate initial infective dose.

Fold up−regulation of Tenecin 1

2.6. Persistence of infection

300

in a plate-reader (BioTek), OD600 measured every 15 minutes and data recorded using Gen5 Data Analysis Software (BioTek). Inoculates were plated out and enumerated as described above. This was repeated 3 times for each bacterial mutant.

50

4

RN4220WT

ΔdltABCD

ptagO G152A

100%

100%

24%

+

−

+

2.7. Statistical analyses WTA:

All statistical analyses were conducted using R (version 2.15.1). Unless otherwise stated, all models that were fit to the data were built first including all possible higher order interactions, and model reduction subsequently performed via likelihood ratio testing (LRT). Highest order non-significant interactions were removed first. A detailed description of the statistical analysis performed can be found in the supplementary material. 2.7.1. Differential recognition of bacterial strains in vivo Fold up-regulation of Tenecin 1 was calculated relative to ribosomal protein rpl27a as the exponent of the difference in the Ct value (threshold PCR cycle for detection of the transcript) between Tenecin 1 and the rpl27a; ΔΔCt method (Schmittgen and Livak, 2008). Fold up-regulation data were analysed with a generalised linear model (see supplement). 2.7.2. Resistance to haemolymph-mediated killing The number of CFU killed was calculated by subtracting the number of CFU remaining after exposure to haemolymph from the initial inoculate, and expressed relative to the control. Data were analysed using generalised linear models (see supplement). 2.7.3. Resistance to CAMPs In order to capture differences in the growth rates of strains in the measurement of resistance to CAMPs, the R package grofit (Kahm et al., 2010) was used to generate MIC50 values. MIC50 was generated to derive a more quantitative and objective measure of resistance than a single visual endpoint measurement such as MIC (see supplement). MIC50 data were log transformed and analysed using general linear models. 2.7.4. Persistence of S. aureus, in vivo CFU persistence data were analysed as raw count data. PBS injected control beetles consistently returned no CFU and were excluded from analysis. All data were analysed using generalised linear models (see supplement). 3. Results 3.1. Recognition by the immune system S. aureus strains appeared to be differentially recognised, as measured by up-regulation of Tenecin 1 in infected insects, but there was no influence of inoculate density. The analysis was therefore

D−alanylation:

Fig. 1. Staphylococcus aureus mutants expressing lower levels of wall teichoic acid are better recognised by the host immune system. Beetles were injected with S. aureus cultures (RN4220WT; n = 23, ptagOG152A; n = 29, ΔdltABCD; n = 25), and mRNA levels of Tenecin 1 were assayed 24 h later as a proxy for immune recognition, relative to the internal control gene rpl27a. Data presented here are representative of all beetles pooled across inoculate densities for each strain. ptagOG152A (24% WTA, D-alanylation positive) bacteria stimulated a significantly higher level of AMP production than either RN4220WT (100% WTA, D-alanylation positive) or ΔdltABCD (100% WTA, D-alanylation negative) (analysis of deviance; p = 0.00119, Tukey multiple-comparison tests; RN4220WT : ptagOG152A; p = 0.0108, RN4220WT : ptagOG152A; p = 0.00354), suggesting they were more readily recognised by host PRRs such as PGRP-SA. RN4220WT and ΔdltABCD were statistically indistinguishable from one another, suggesting that recognition of bacteria is not enhanced in the absence of D-alanylation of WTA (Tukey multiple comparison test; WT : dltABCD; p = 0.961). Data are given ± 1 standard error and bars bearing the same symbol were statistically indistinguishable.

simplified by pooling data across inoculate densities for each strain. The pooled data show that reduced WTA (ptagOG152A) mutant elicits a significantly higher immune response than either the D-alanylation mutant (ΔdltABCD) or the parental strain (RN4220WT), which were statistically indistinguishable from one another (analysis of deviance; χ2 = 13.4, df = 2, p = 0.00119, Fig. 1). No mortality was observed in the 24 h following infection for this experiment. 3.2. Immuno-resistance against haemolymph and AMPs Both density of WTA at the cell surface and D-alanylation of teichoic acids influence bacterial survival upon exposure to immuneactivated host haemolymph (analysis of deviance; χ2 = 86.779, df = 10, p < 0.001, Fig. 2). Low-density WTA mutants were more susceptible to killing by haemolymph than parental RN4220 (Fig. 2). In contrast, the low-density WTA mutants (ptagOG152A and pMAD, 24% and 0% WTA respectively) displayed only marginally higher susceptibility to isolated AMPs, as measured by MIC50 analysis (Table 2) and on multiple-comparison testing, no differences in susceptibility between strains were resolved (Tukey multiple-comparison tests; p > 0.05). The D-alanylation mutant (ΔdltABCD) was necessarily excluded from this analysis as MIC50 values could not be generated. However, it is clear that the D-alanylation mutant is far more susceptible to CAMP mediated killing than any of the other strains, exhibiting > 64 times lower resistance to Tenecin 1 than all other strains, and >8 and >16 times lower resistance to pexiganan than the parental strain (RN4220WT) and the WTA depleted ΔtagO mutants

Please cite this article in press as: Jack Dorling, Caroline Moraes, Jens Rolff, Recognition, survival and persistence of Staphylococcus aureus in the model host Tenebrio molitor, Developmental and Comparative Immunology (2014), doi: 10.1016/j.dci.2014.08.010

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Table 2 Resistance of Staphylococcus aureus wall teichoic acid and D-alanylation deficient mutants to cationic AMPs. Resistance to CAMPs recorded as concentration required to halt growth completely (MIC), or reduce growth rate (r0) by 50% (MIC50). CAMP Tenecin 1

MIC MIC50 MIC MIC50 MIC MIC50

Pexiganan Cercropin-A

RN4220WT

ΔdltABCD

Strain: ptagO

ptagOG152A

pMAD

16 9.39 ± 1.13 2 0.85 ± 0.0108 >32 nd

32 nd

16 7.57 ± 1.23 4 1.01 ± 0.0118 >32 nd

16 7.61 ± 0.291 4 1.09 ± 0.0279 >32 nd

All MIC and MIC50 concentrations are given in μg mL−1; nd – not determined.

50

100

δ α

α

1e+08

RN4220WT ΔdltABCD ptagO G152A PBS

1e+05

δ

Our study provides fresh insight into infection dynamics of S. aureus, in relation to the density of WTA expressed at the cell surface, and the D-alanylation of this peptidoglycan-concealing polysaccharide coat. In studying the resistance and persistence of these

100

200 150

β

4. Discussion

WTA: D−alanylation:

RN4220WT

ΔdltABCD

ptagO

ptagO G152A

pMAD

100%

100%

90%

24%

0%

+

−

+

+

+

0

0

CFU x 103 killed relative to control

As previously observed (Haine et al., 2008a) parental strain S. aureus was able to persist up to 10 days, although this time in male insects. D-alanylation deficient S. aureus however, was cleared more rapidly while the lower density WTA mutant (ptagOG152A) was cleared at a seemingly intermediate rate (analysis of deviance; χ2 = 34.643, df = 2, p < 0.001, Fig. 3) and at 10 days, no insects remained infected. All strains did however display a significant decline in CFU over time (analysis of deviance; χ2 = 72.962, df = 1, p < 0.001,

0.1

3.3. Persistence of infection

Fig. 3). Additionally, there was a significant negative effect of insect wet body mass on persistence (analysis of deviance; χ2 = 8.658, df = 1, p = 0.00325), but this did not differ between the different strain treatments (ANOVA; F = 0.6577, df = 2, 75, p = 0.521), and no interaction of any modelled variables was observed. Some mortality was observed during this experiment (Day 10: RN4220WT; 41%, ΔdltABCD; 0%, ptagOG152A; 9%, PBS; 25%) and consequently these beetles were censored from any analysis.

Number of CFU in haemolymph*

(ptagO, ptagOG152A and pMAD) respectively. MIC data suggest that the ΔtagO mutants may in fact show some increased resistance to pexiganan mediated killing relative RN4220WT (Table 2) although this is not reflected in the results of the MIC50 analysis.

0

2

4

6

8

10

Time post−infection / days* Fig. 2. Both Staphylococcus aureus mutants expressing lower levels of WTA and deficient in the ability to D-alanylate WTA were significantly more susceptible to killing by extracted host haemolymph. S. aureus strains were exposed to haemolymph extracted from beetles initially challenged with ~5 × 106 CFU of RN4220WT mixed with anitcoagulant buffer. Control bacteria were exposed to only anticoagulant buffer. Significant differences in bacterial killing by host haemolymph were observed (analysis of deviance; p < 0.001). ΔdltABCD (100% WTA, D-alanylation negative) bacteria were killed significantly more than any of the other strains (Tukey multiple comparison tests; RN4220WT : ΔdltABCD; p < 0.001, ptagOG152A : ΔdltABCD; 0.00334, pMAD : ΔdltABCD; p < 0.001) likely underlain by AMPs present in the host haemolymph. Additionally, mutants depleted in WTA content (ptagOG152A; 24% WTA, D-alanylation positive, pMAD; 0% WTA, D-alanylation positive) were killed significantly more than either RN4220 W T (100% WTA, D-alanylation positive) or ptagO (90% WTA, D-alanylation positive) (Tukey multiple comparison tests; RN4220WT : ptagOG152A; p < 0.001, ptagO : ptagOG152A ; < 0.001, RN4220 WT : pMAD; p < 0.0417, ptagO : pMAD; < 0.0166), suggesting that some other components of the host humoral immune system such as lysozyme production or the melanisation cascade may be involved in killing of bacteria depleted for WTA. Data are given ± 1 standard error and bars bearing the same symbol were statistically indistinguishable. All experiments were repeated in triplicate and average values are presented here.

Fig. 3. Staphylococcus aureus strains depleted for WTA and impaired in the ability to D-alanylate WTA show reduced ability to persist within the host relative to parental strain bacteria. Beetles were injected with ~5 × 106 CFU of either RN4220WT (n = 34), ΔdltABCD (n = 32), ptagOG152A (n = 32) or PBS (n = 32) and bacteria were harvested at 1, 3, 7 and 10 days post infection and enumerated. All strains declined in persistence over time (analysis of deviance; p < 0.001) and showed significant differences in their ability to persist (analysis of deviance; p < 0.001). RN4220WT and ptagOG152A persisted at a higher level than ΔdltABCD (Tukey multiple comparison tests; RN4220WT : ΔdltABCD; p < 0.001, ptagOG152A : ΔdltABCD; p < 0.001), although RN4220WT and ptagOG152A were apparently statistically indistinguishable from one another (RN4220WT : ptagOG152A; p = 0.521) despite clearance of ptagOG152A by all insects assayed at the end of the experiment. This suggests that in fact, both of the mutant strains were impaired in their ability to persist within the host, likely due to the differences in recognition and in vitro killing of these strains seen. Data are given ± 1 standard error, and PBS baseline is included to demonstrate sterility of the haemolymph of these beetles. Each data point represents 4–11 beetles as some mortality was observed (Day 10: RN4220WT; 41%, ΔdltABCD; 0%, ptagOG152A; 9%, PBS; 25%). The initial points on the graph are derived from plating of the initial inoculate, not from beetles injected at time 0 h.

Please cite this article in press as: Jack Dorling, Caroline Moraes, Jens Rolff, Recognition, survival and persistence of Staphylococcus aureus in the model host Tenebrio molitor, Developmental and Comparative Immunology (2014), doi: 10.1016/j.dci.2014.08.010

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J. Dorling et al./Developmental and Comparative Immunology ■■ (2014) ■■–■■

strains alongside recognition in a single infection model, our results help to build a more complete picture of the multi-faceted function of these key bacterial molecules during infection. WTA expression modulated immune-recognition of bacteria by the host and bacterial immuno-resistance to crude haemolymph, with D-alanylation of this glycopolymer further influencing the latter of these traits. We propose that these properties may ultimately underlie persistence of S. aureus in this system. In the absence of WTA recognition by the host was increased, suggesting a role for WTA in limiting recognition of PGN by host PGRPs such as PGRP-SA. Additionally, the impairment of bacteria to D-alanylate WTA significantly decreased resistance of S. aureus to both isolated cAMPs and to the pooled humoral immune responses of T. molitor. The data also clearly show that absence of D-alanylation significantly decreased the ability of S. aureus to persist during infection. Taken together, these results implicate WTA and its D-alanylation as important components in the establishment and maintenance of staphylococcal infection. This also highlights that complexities at the bacterial cell surface in the same bacterial strain background can lead to specific outcomes of infection. A study by Atilano et al. (2010) used the same strains as in this study and PGRP knock-down flies (D. melanogaster) to assess the role of recognition in the pathogenicity of the various strains. In agreement with our results, WTA expression impaired recognition of the bacteria, as measured by binding of PGRP-SA to the bacterial cell surface. We have also shown that this mutant upregulates downstream AMP expression, likely as a result of such recognition by PGRP-SA in T. molitor. This is however in contrast to the results of Kurokawa et al. (2011), who documented increased recognition of D-alanylation deficient mutants. However, this could be due to incomplete removal of proteins or lipoteichoic acids in WTA bound PGN preparations, rather than recognition of WTA itself, which is not currently known to be immunogenic. PGRP-SA alone has been shown to reconstitute recognition via the T. molitor Toll-pathway in vitro (Kim et al., 2008; Kurokawa et al., 2011) This suggests that in T. molitor recognition of S. aureus may be quite different from that of D. melanogaster, as PGRP-SD is required to enhance binding of PGRP-SA to D-alanylated WTA bound PGN in D. melanogaster (Wang et al., 2008). A recent RNAseq experiment produced for S. aureus infection of T. molitor also did not yield any homologues of PGRP-SD (Johnston et al., 2014). Atilano et al. (2011) also suggest that reduced pathogenicity of strains lacking D-alanylation is due to some other mechanism than increased recognition, in agreement with our results. D-alanylation deficient strains were still handicapped during infection of D. melanogaster lacking either PGRP-SA or PGRP-SD respectively, in contrast to independently tested ΔtagO mutants (Atilano et al., 2010). Although it is possible that in the absence of D-alanylation not all of these D. melanogaster PGRPs are required for recognition of PGN, and their individual functions may be flexible (Wang et al., 2008; Wang and Beerntsen, 2013) this is more likely underlain by the susceptibility of this mutant to killing by AMPs in the haemolymph of these insects, as seen in our study. D-alanylation is a non-essential modification of WTA (Li et al., 2007) and so there is likely little phenotypic cost outside of resistance to CAMPs for the abolition of D-alanylation. In fact, D-alanylation is up-regulated in response to challenge by antimicrobial peptides, with the dltABCD operon (responsible for D-alanylation and absent from strain dltABCD) being stimulated by a regulatory system known as ApsXRS (Li et al., 2007). This further strengthens the conclusions that D-alanylation could be a dedicated process in resistance to the inducible AMP response, and a much stronger candidate in mediating infection persistence than WTA itself, which is likely more involved in evasion of immune recognition.

Surprisingly, there has been little work on investigating the relevant mechanisms by which WTA may influence long term infection. Many Gram-positive pathogens such as S. aureus are known to cause persistent, long lasting infections (Foster, 2005) and this includes several important human pathogens such as S. aureus (Li et al., 2012). In vitro evidence has also implicated D-alanylation in resistance to cationic AMPs (CAMPs) due to its influence on cell surface charge (Peschel and Sahl, 2006). Thus, the results presented here represent first steps into investigating the role of D-alanylation in longlasting infections, where prolonged exposure to host immune effectors such as CAMPS is highly likely to occur. Here we found that the D-alanylation deficient mutant (dltABCD) persists within the host at least up to 3 days post-infection but at 10 days was cleared from all but one host while RN4220WT persisted at a much higher level. This is of particular significance when considered in conjunction with the demonstration here of drastically reduced resistance of ΔdltABCD to CAMPs and the host humoral immune response. Persistence for this amount of time is likely concurrent with prolonged exposure to AMPs, mRNA expression of which is known to be strongly up-regulated for at least 7 days (Johnston et al., 2014), peaking after 24 h and remaining at high levels. Similarly, haemolymph antimicrobial activity increases in a near identical pattern (Haine et al., 2008a, 2008b). Taken together, these results strongly suggest that D-alanylation mediated immunoresistance to CAMPs is likely to be playing a role in maintaining persistent infection in this system. Significant differences were also found between ptagOG152A and RN4220 in persistence, and by day 10 no beetles remained infected with ptagOG152A, showing that this mutant is impaired in its ability to persist relative to RN4220WT. Indeed ptagOG152A and pMAD (24% and 0% WTA respectively) were also more susceptible to killing by haemolymph of T. molitor relative to RN4220WT and ptagO. This suggests that reduced WTA expression increases susceptibility to host immune effectors, and may explain at least in part the clearance of ptagOG152A from the haemolymph of T. molitor, in combination with the increased stimulation of the humoral immune system by this mutant, including components such as the melanisation cascade (Park et al., 2006). Additionally, in vivo, bacteria will be exposed to a cocktail of AMPs, rather than just single cAMPs in isolation (Johnston et al., 2014) and this may possibly explain why WTA depletion mutants showed decreased resistance to a pooled host response (haemolymph) relative to single effectors. It should be noted however, that although the patterns here are consistent with either immune evasion or immuno-resistance mediated persistence, it is difficult to disentangle the relative contributions of these different processes in determining persistence. What this does reveal however, is that trade-offs in WTA acid expression may in part contribute to specific outcomes in this infection model. Interestingly, we also show here the persistence of these bacteria in male insects, although with mortality of insects which was not previously noted (Haine et al., 2008a). This could potentially be due to sexual dimorphism in immunity in this insect (Rolff et al., 2005), a potential further contributor to specificity of interactions between host and parasite within a population. Previous study has documented increased immuno-resistance of S. aureus during persistent infection (Haine et al., 2008a) suggesting immuno-resistance may be more important than initial evasion of the immune system. The data in this study outline the potential importance of WTA expression and its D-alanylation in persistent infection. Combined with the knowledge that D-alanylation is up-regulated upon exposure to AMPs this presents the hypothesis that a phenotypically plastic response involving the up-regulation of D-alanylation upon exposure to cAMPs (Li et al., 2007) may, at least in part, underlie increases in immuno-resistance as observed in Haine et al. (2008a). Additionally, depletion of

Please cite this article in press as: Jack Dorling, Caroline Moraes, Jens Rolff, Recognition, survival and persistence of Staphylococcus aureus in the model host Tenebrio molitor, Developmental and Comparative Immunology (2014), doi: 10.1016/j.dci.2014.08.010

ARTICLE IN PRESS J. Dorling et al./Developmental and Comparative Immunology ■■ (2014) ■■–■■

WTA has been shown to impair nasal colonisation of a rat host (Weidenmaier et al., 2004), is virulence attenuated in mice (Collins et al., 2002) and has decreased pathogenicity in a mouse model of endophthalmitis (Suzuki et al., 2011). This suggests, along with the data of Atilano et al. (2011), a potential mechanism for this finding. Depletion of WTA could be increasing exposure of PGN to the host immune system and importantly, modification of WTA with D-alanine could be required for resistance to downstream immune effectors produced during these infections, such as AMPs. Acknowledgements We would like to thank Petros Ligoxygakis and Sergio Filipe for providing the S. aureus strains and Paul Johnston for help and discussion. C.M. was funded by a fellowship from CNPq/INCT, Brazil to Fernando Ariel Genta. The research was supported by European Research Council grant 260986 (to J.R.). Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.dci.2014.08.010. References Armitage, S.A.O., Peuß, R., Kurtz, J., 2014. Dscam and pancrustacean immune memory – a review of the evidence. Dev. Comp. Immunol. doi:10.1016/j.dci.2014.03.004; in press. Atilano, M.L., Pereira, P.M., Yates, J., Reed, P., Veiga, H., Pinho, M.G., et al., 2010. Teichoic acids are temporal and spatial regulators of peptidoglycan cross-linking in Staphylococcus aureus. Proc. Natl. Acad. Sci. U.S.A. 107, 18991–18996. doi:10.1073/ pnas.1004304107. Atilano, M.L., Yates, J., Glittenberg, M., Filipe, S.R., Ligoxygakis, P., 2011. Wall teichoic acids of Staphylococcus aureus limit recognition by the Drosophila peptidoglycan recognition protein-SA to promote pathogenicity. PLoS Pathog. 7, e1002421. doi:10.1371/journal.ppat.1002421. Bera, A., Biswas, R., Herbert, S., Kulauzovic, E., Weidenmaier, C., Peschel, A., et al., 2007. Influence of wall teichoic acid on lysozyme resistance in Staphylococcus aureus. J. Bacteriol. 189, 280–283. doi:10.1128/JB.01221-06. Chae, J.-H., Kurokawa, K., So, Y.-I., Hwang, H.O., Kim, M.-S., Park, J.-W., et al., 2012. Purification and characterization of tenecin 4, a new anti-Gram-negative bacterial peptide, from the beetle Tenebrio molitor. Dev. Comp. Immunol. 36, 540–546. doi:10.1016/j.dci.2011.09.010. Collins, L.V., Kristian, S.A., Weidenmaier, C., Faigle, M., Van Kessel, K.P.M., Van Strijp, J.A.G., et al., 2002. Staphylococcus aureus strains lacking D-alanine modifications of teichoic acids are highly susceptible to human neutrophil killing and are virulence attenuated in mice. J. Infect. Dis. 186, 214–219. doi:10.1086/341454. Dong, Y., Taylor, H.E., Dimopoulos, G., 2006. AgDscam, a hypervariable immunoglobulin domain-containing receptor of the Anopheles gambiae innate immune system. PLoS Biol. 4, e229. doi:10.1371/journal.pbio.0040229. Foster, T.J., 2005. Immune evasion by staphylococci. Nat. Rev. Microbiol. 3, 948–958. doi:10.1038/nrmicro1289. Geissmann, Q., 2013. OpenCFU, a new free and open-source software to count cell colonies and other circular objects. PLoS ONE 8, e54072. doi:10.1371/ journal.pone.0054072. Haine, E.R., Moret, Y., Siva-Jothy, M.T., Rolff, J., 2008a. Antimicrobial defence and persistent infection in insects. Science 80, 322, 1257–1259. Haine, E.R., Pollitt, L.C., Moret, Y., Siva-Jothy, M.T., Rolff, J., 2008b. Temporal patterns in immune responses to a range of microbial insults (Tenebrio molitor). J. Insect Physiol. 54, 1090–1097. doi:10.1016/j.jinsphys.2008.04.013. Johnston, P.R., Makarova, O., Rolff, J., 2014. Inducible defenses stay up late: temporal patterns of immune gene expression in Tenebrio molitor. G3 4, 947–955. doi:10.1534/g3.113.008516.

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Please cite this article in press as: Jack Dorling, Caroline Moraes, Jens Rolff, Recognition, survival and persistence of Staphylococcus aureus in the model host Tenebrio molitor, Developmental and Comparative Immunology (2014), doi: 10.1016/j.dci.2014.08.010