ExsB is required for correct assembly of Pseudomonas aeruginosa type III secretion apparatus in ...

39 Pages·2015·0.89 MB·English

IAI Accepted Manuscript Posted Online 17 February 2015
Infect. Immun. doi:10.1128/IAI.00048-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
ExsB is a T3SS pilotin
1 ExsB is required for correct assembly of Pseudomonas aeruginosa type III
2 secretion apparatus in the bacterial membrane and full virulence in vivo
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4 Running title: ExsB is a T3SS pilotin
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6 Caroline Perdu1,2,3,4, *, Philippe Huber1,2,3,4, Stéphanie Bouillot1,2,3,4, Ariel Blocker5, Sylvie w
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7 Elsen1,2,3,4, Ina Attrée1,2,3,4, Eric Faudry1,2,3,4, # a
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9 1 Univ. Grenoble Alpes, Bacterial Pathogenesis and Cellular Responses Group, F-38000, m

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10 Grenoble, France p
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11 2 CEA, iRTSV, F-38000, Grenoble, France iai.
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12 3 CNRS, ERL5261, F-38000, Grenoble, France m
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13 4 INSERM, U1036_S Biology of Cancer and Infection Laboratory, F-38000 Grenoble, France rg
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14 5 Schools of Cellular & Molecular Medicine and Biochemistry, University of Bristol, Bristol, n
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15 BS8 1TD, United Kingdom ril
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16 * Present address: TWB - LISBP- INSA, 135 avenue de Rangueil, 31077 Toulouse Cedex 4 ,

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17 # Corresponding author: [email protected] 1
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ExsB is a T3SS pilotin
19 ABSTRACT
20 Pseudomonas aeruginosa is responsible for high morbidity infections of cystic fibrosis patients
21 and is a major agent of nosocomial infections. One of its most potent virulence factors is a Type
22 III Secretion System (T3SS) that injects toxins directly into the host cell cytoplasm. ExsB, a
23 lipoprotein localized in the bacterial outer membrane, is one of the components of this
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24 machinery, of which the function remained elusive until now. The localization of the exsB gene o
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25 within the exsCEBA regulatory gene operon suggested an implication in the T3SS regulation, lo
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26 while its similarity with yscW from Yersinia spp argued in favor of a role in machinery assembly. d

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27 The present work shows that ExsB is necessary for full in vivo virulence of P. aeruginosa. o
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28 Furthermore, the requirement of ExsB for optimal T3SS assembly and activity is demonstrated t
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29 using eukaryotic cell infection and in vitro assays. In particular, ExsB promotes the assembly of /ia
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30 the T3SS secretin in the bacterial outer membrane, highlighting the molecular role of ExsB as a s
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31 pilotin. This involvement in the regulation of the T3S apparatus assembly may explain the r
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32 localization of the ExsB-encoding gene within the regulatory gene operon. o
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33 p
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34 1
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38 Keywords: Type III Secretion, virulence, infection, Pseudomonas aeruginosa, pilotin, secretin
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39 INTRODUCTION
40
41 Pseudomonas aeruginosa is a gram negative bacterium that thrives in a variety of
42 environments (1, 2) and can colonize diverse hosts, from invertebrates to humans (3, 4 ). It
43 represents a real threat to human health, being responsible for the most frequent hospital-acquired
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44 infections along with Escherichia coli and Staphylococcus aureus in France (5). Moreover, it is o
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45 the first cause of mortality and morbidity for people suffering from cystic fibrosis (6). As most lo
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46 clinical isolates are multiresistant to antibiotics, it is imperative to find new anti-bacterial d

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47 strategies against this bacterium (7, 8). In this perspective, a better understanding of the virulence o
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48 mechanisms of P. aeruginosa turned out to be a valuable approach to identify therapeutic targets t
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49 and eventually develop new and more specific drugs. /ia
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50 One major virulence factor of P. aeruginosa is the Type III Secretion System (T3SS). s
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51 This system is well conserved among bacterial pathogens, such as Yersinia pestis, the causative r
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52 agent of plague, and Salmonella species or Shigella flexneri, responsible for intestinal diseases o
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53 (9). It has been shown that the T3SS of P. aeruginosa is particularly active during acute p
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54 infections and is associated with poor clinical outcomes (10). Indeed, the presence of active T3SS 1
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55 is correlated with an increased mortality (11). 0
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56 The T3SS allows the bacterium to inject toxins directly into the host cell cytoplasm in b
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57 order to hijack several cellular pathways, leading to disruption of the actin cytoskeleton and to u
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58 cell death. This injection machinery consists of more than twenty proteins, assembled in a
59 syringe-like structure and composed of three complexes: the basal body, the needle and the
60 translocon (12). In the cytoplasm, a proteinaceous ring makes the connection between the basal
61 body and other soluble proteins. The basal body spans the two bacterial membranes and is made
62 of three major proteins: PscD and PscJ in the inner membrane and the secretin PscC in the outer
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63 membrane. Structural studies on T3SS from different bacteria indicate that the outer membrane
64 components oligomerize into rings of 12 or 15 subunits while the inner membrane proteins form
65 24-mer assemblies (13). The needle is anchored to the basal body and forms a 80 nm long narrow
66 channel that protrudes from the bacterial surface. It is assembled by polymerization of the small
67 protein PscF. Finally, the translocon, consisting of the tip protein PcrV and the pore proteins
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68 PopB and PopD, forms a pore at the extremity of the needle, into the eukaryotic membrane (14). o
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69 The presence of the secretin ring in the outer membrane is essential for the T3SS activity. lo
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70 Proteins belonging to the secretin family are found in several protein transport systems, such as d

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71 the Type II Secretion System or the Type IV pili (15). T3SS secretins assemble in oligomeric o
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72 rings of 12 or 15 subunits, with a 5 nm diameter pore, that were observed by electron microscopy t
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73 (16-22). The T3SS secretin oligomers are very stable, heat- and SDS-resistant, and have a high /ia
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74 molecular weight of 985 kDa in Y. enterocolitica (17). The N-terminal domains of secretins s
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75 display low similarity and are proposed to have a role in specific interactions in each system. On r
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76 the opposite, the C-terminal domains are conserved and involved in the oligomerization and pore o
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77 formation. However, a small non-conserved stretch at their C-terminal end was found, in some p
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78 studies, to interact with small lipoproteins, named pilotins (23, 24). Indeed, the secretins often 1
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79 require other proteins, in particular pilotins, for a correct targeting and/or assembly in the outer 0
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80 membrane (25). b
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81 Pilotins are usually low-molecular weight lipoproteins that were previously classified into u
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82 three structural classes (25), before a fourth class was more recently characterized (26). The study
83 of some pilotins from different secretion systems showed that they are involved in the stability,
84 localization and/or oligomerization of secretins. For instance, the well-studied pilotins OutS from
85 the T2SS of Dickeya dadantii, PilW and PilF from the Type IV pili of Neisseria sp. and P.
86 aeruginosa, respectively, were shown to be required for the localization in the outer membrane
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87 and/or assembly and/or stability of the oligomers of OutD and PilQ secretins (27-30). Some T3SS
88 pilotins were also characterized. In the case of S. flexneri, two lipoproteins, MxiM and MxiJ,
89 participate to secretin oligomerization and stabilization (31-33). In S. typhimurium and Y.
90 enterocolitica, the deletions of the lipoproteins genes invH and yscW, respectively, lead to a
91 decreased quantity of secretin oligomers InvG and YscC, respectively, and a mislocalization of
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92 the secretin to the inner membrane instead of the outer membrane (34, 35). Moreover, a o
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93 phenotypic analysis of Yersinia and Salmonella strains lacking the YscW or InvH pilotin revealed lo
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94 a drastic decrease in protein secretion as well as in apoptosis of infected cells (36-40). d

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95 The ExsB protein from Pseudomonas aeruginosa displays 26% sequence identity with the o
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96 YscW pilotin and is also predicted to be a lipoprotein with a low molecular weight of 15.6 kDa t
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97 and a high isoelectric point of 9.4 (36, 41). It was thought for more than a decade that this protein /ia
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98 was not produced in P. aeruginosa (42), suggesting an important evolutionary divergence s
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99 between P. aeruginosa and Yersinia spp (35). However, our previous work indicated that ExsB is r
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100 expressed and located in the bacterial outer membrane and its three dimensional structure was o
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101 determined. It folds in seven β-strands and one α-helix and exhibits two charged zones that could p
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102 be involved in the interaction with lipids and other proteins (41). Nevertheless, this fold was 1
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103 functionally uninformative, especially since pilotins display very different ones (25, 26). 0
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104 Moreover, the role of ExsB in P. aeruginosa was not investigated previously, due to the position b
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105 of the gene within the regulatory gene operon that makes genetic manipulations difficult. Indeed, u
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106 its gene is localized immediately downstream from exsE, encoding a component of the T3SS
107 regulatory cascade, and upstream from the gene encoding ExsA, the main transcriptional
108 activator of the system.
109 The aim of this study was to establish the role of ExsB in P. aeruginosa general toxicity
110 and more specifically in the functioning of its T3SS. In this perspective, a comparative study of
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111 the phenotypes of a mutant strain lacking exsB gene, the corresponding complemented strain and
112 the wild-type strain was carried out. In vivo and in vitro analyses demonstrated the requirement of
113 ExsB for P. aeruginosa complete virulence and for T3SS optimal activity. Moreover, the
114 investigation of the molecular role of ExsB revealed its role in the secretin assembly.
115
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116 MATERIALS & METHODS o
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117 lo
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118 Bacterial strains, plasmids, and growth conditions d

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119 Bacterial cultures were grown in Luria-Bertani (LB) broth at 37°C and 300 rpm. The o
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120 antibiotics were added at the appropriate concentrations: 300 mg/L for carbenicillin, 25 mg/L for t
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121 kanamycin and 10 mg/L for tetracycline. For in vitro T3SS induction in P. aeruginosa strains, the /ia
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122 culture medium was depleted in calcium with 5 mM EGTA and 20 mM MgCl and bacterial s
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123 suspensions were used at an optical density (OD600) of 1, which corresponds to the exponential r
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124 growth phase. o
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125 The exsB deletion mutant, CHAΔexsB, was constructed in several steps with Splicing p
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126 Overlapping Extension (SOE)-PCR. Briefly, the two flanking regions of exsB were amplified 1
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127 from the pIA60 plasmid (see Table 1) with the primer pairs Delta-exsB_1/Delta_ExsB_2 (see 0
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128 Table 2), and Delta_ExsB_3/Delta_ExsB_4, respectively. The two resulting DNA fragments b
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129 were used together for another PCR step with the primers Delta_exsB_1 and Delta_exsB_4,
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130 leading to a DNA fragment containing only 4% of exsB sequence
131 (GTGAGGTGCTGGATGTGA). Then this DNA fragment was cloned into the SmaI restriction
132 sites of the pEX100T suicide plasmid containing the counter-selectable sacB marker. The
133 plasmid was introduced into P. aeruginosa CHA strain by triparental mating, using the
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134 conjugative properties of the helper plasmid pRK2013. Co-integration events were selected on
135 PIA plates containing carbenicillin. Single colonies were then plated on PIA medium containing
136 5% sucrose to select for the loss of plasmid: the resulting strains were checked for carbenicillin
137 sensitivity and the gene deletion was confirmed by PCR.
138 The DNA fragments needed for introducing a TGA codon in the exsB sequence were
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139 generated by SOE-PCR. A 295 bp fragment encompassing the upstream and 5’ region of exsB o
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140 was amplified using CHA genomic DNA and primers ExsB-TGA-H-F1 (EcoRI site at its lo
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141 5’)/ExsB-TGA-H-R1. The ExsB-TGA-H-F2/ ExsB-TGA-H-R2 (BamHI site at its 5’) were used d

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142 to amplify the downstream 337 bp fragment of exsB. The overlapping primers generated a o
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143 modification of the 12th codon, replacing TGC by a TGA stop codon, and converted the t
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144 downstream GTCAGC sequence into a GTCGAC HindII site helpful for further mutant /ia
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145 identification. The two fragments were used for a third PCR step using ExsB-TGA-H-F1/ExsB- s
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146 TGA-H-R2 primers. The resulting 602 bp fragment was cloned into pCR-Blunt II-TOPO vector r
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147 and sequenced. After cleavage with EcoRI-BamHI, the SOE-PCR fragment was inserted into the o
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148 suicide plasmid pEXG2 (containing sacB) giving pEX-ExsB TGA-H. The plasmid was p
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149 introduced into P. aeruginosa CHA strain by triparental mating as described above, using 1
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150 gentamycin instead of carbenicillin. The mutation was first identified by the presence of HindII 0
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151 site inserted contiguous to the TGA stop codon, then confirmed by sequencing of the CHA-B b
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152 strain. u
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153 For the chromosomic complementation of the mutants, exsB gene with its own RBS was
154 amplified and inserted into the pIApC plasmid using the restriction sites XbaI and HindIII. The
155 resulting DNA fragment containing exsB fused to pC, the promoter of the exsCEBA operon, was
156 then extracted and cloned into the miniCTX integrative vector. Finally, the complemented strains
157 CHAΔexsB/exsB and CHA-B /exsB were obtained after transfer of the construct into the
TGA
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158 mutant strains, transformation with the pFLP2 plasmid to excise the miniCTX backbone and
159 selection on media containing 5% sucrose to select for the loss of pFLP2.
160
161 Electrophoresis and western blot
162 T3SS-induced cultures of P. aeruginosa (1 ml) were centrifuged at 13 000 g for 5 minutes to
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163 separate cells from supernatants. Cells were resuspended in 100 µl of Laemmli loading buffer 1X o
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164 to measure the levels of intracellular PopB, PcrV, ExsB and PscC while 100 µl of supernatants lo
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165 were mixed with 25 µl of Laemmli loading buffer 5X to monitor secreted PopB and PcrV. ExsA, d

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166 ExsD and PscG, were detected in soluble extracts corresponding to 50X cell suspensions lysed by o
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167 sonication and cleared from membrane and cellular debris by ultracentrifugation. Proteins were t
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168 separated by SDS-PAGE, using 15% or 18% acrylamide gels, and transferred onto nitrocellulose /ia
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169 membranes. For the analysis of secretin oligomers, 8% acrylamide gels were used. The s
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170 membranes were saturated in non-fat milk 5% and incubated with primary antibodies raised r
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171 against PopB (43), ExsB (41), PcrV (44), PscC (our laboratory), ExsA and ExsD (45), PscG (46), o
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172 XcpY (kind gift of R. Voulhoux), RpoA (from NeoClone), and OprI-F (given by G.E. Gilleland). p
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173 Secondary horseradish-peroxidase-conjugated antibodies were then added, followed by 1
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174 chemiluminescence detection (LuminataTM Classico, Millipore) 0
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175 b
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176 Mice infection u
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177 For each P. aeruginosa strain, ten BALB/C mice (males weighting 20g, from Harlan breeding)
178 were infected by inhalation of 2x106 bacteria in order to induce pneumopathy. The survival was
179 followed every 2 hours. For the statistical analysis, Kaplan Meyer curves were established and
180 the Log Rank test was applied.
181
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182 Cell retraction assay
183 HUVEC were isolated from human umbilical cord and cultured at 37°C, 5% CO in EBM2
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184 medium containing 2% of fetal bovine serum. The infection was set up at a MOI of 1 by P.
185 aeruginosa strains at an OD of 1. As formerly described (47), cells were fixed with cold
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186 methanol at 3 hours post-infection. They were then labeled with a β-actin antibody and with a
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187 fluorescent secondary antibody. In these conditions, the primary antibody detects free actin along w
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188 with actin fibers thus providing a uniform cell staining. For each condition, the field surface lo
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189 covered by the cells on three distinct pictures was measured with the ImageJ software. The d

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190 percentage of cellular retraction was calculated by normalizing the infected conditions to the non- m

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191 infected condition. One-way ANOVA with Tukey post-hoc test was performed to compare the tt
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192 mean percentages of cellular retraction. ia
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193 s
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194 Injection of chimeric toxin r
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195 The injection of the chimeric toxin ExoS-β-lactamase was assayed as previously described (48). on

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196 BJAB cells were grown at 37°C, 5% CO2 in RPMI medium containing 10% of fetal bovine ril
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197 serum. After 3 hours of infection by P. aeruginosa strains producing the chimeric toxin, BJAB 1
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198 cells were incubated with 1mM CCF2/AM (Invitrogen) during 30 min and their fluorescence was 1
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199 then analyzed by flow cytometry (FACS MoFlo). The intact CCF2 probe emits a green y

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200 fluorescence but its cleavage by the β-lactamase domain of the chimeric toxin leads to the e
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201 formation of a product emitting a blue fluorescence. Dead cells were removed based on FSC/SSC
202 parameters and the blue fluorescence of the remaining cells was measured. A gate in the
203 histogram plot allows the definition of the highly blue fluorescent population corresponding to
204 the injected cells.
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205 Quantification of T3SS needles on bacterial surface
206 After induction of the T3SS, 500µL of P. aeruginosa suspension was collected and centrifuged at
207 13000g for 5 minutes at 4°C. Bacteria were concentrated 10 times in PBS. 3µL of this suspension
208 was dropped onto glow discharged, copper 200 mesh, carbon and formvar coated grids. After 3
209 washes with filtered water, a negative staining was accomplished by incubation with 1% uranyl
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210 acetate during 1 minute. The samples were observed by electron microscopy, with a FEI Tecnai o
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211 12 fitted with an FEI Eagle 4k by 4k CCD camera at a magnification of 23000 using the FEI lo
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212 Tecnai Imaging Analysis (TIA) software. The T3S needles were identified by their size, with a d

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213 diameter of about 6.5 nm and a length of 70 nm. The number of needles present at the surface of o
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214 the bacteria as well as the detached needles was determined on 60 pictures for both the exsB t
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215 mutant and complemented strains. /ia
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216 s
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217 Inner and outer membrane separation r
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218 Membrane separation was achieved on the three P. aeruginosa strains overexpressing the T3SS o
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219 activator ExsA. A sucrose gradient was used to separate the membranes according to their p
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220 density, as previously described (49). The main steps of this procedure are recapitulated here. 1
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221 First, total membranes from 500 ml culture were prepared in 500µL of a buffer containing 8 mM 0
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222 Tris–HCl at pH 7.4, 20% sucrose (w/v), 4 mM ethylenediaminetetraacetic acid and protease b
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223 inhibitor cocktail complete (Roche). They were then loaded on a 35–55% sucrose gradient and u
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224 submitted to 72 hours of ultracentrifugation at 190 000g. Finally, 15 fractions of 750 µL were
225 collected and analyzed by SDS-PAGE and Western blotting. For illustration, representative
226 fractions of the inner and outer membranes were also analyzed by SDS-PAGE and western
227 blotting. NADH oxidase activity, a marker of inner membranes, was measured as previously
228 described (50).
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