The multifunction Coxiella effector Vice stimulates macropinocytosis and interferes with the ESCRT machinery.

Intracellular bacterial pathogens divert multiple cellular pathways to establish their niche and persist inside their host. Coxiella burnetii, the causative agent of Q fever, secretes bacterial effector proteins via its Type 4 secretion system to generate a Coxiella-containing vacuole (CCV). Manipulation of lipid and protein trafficking by these effectors is essential for bacterial replication and virulence. Here, we have characterized the lipid composition of CCVs and found that the effector Vice interacts with phosphoinositides and membranes enriched in phosphatidylserine and lysobisphosphatidic acid. Remarkably, eukaryotic cells ectopically expressing Vice present compartments that resemble early CCVs in both morphology and composition. We found that the biogenesis of these compartments relies on the double function of Vice. The effector protein initially localizes at the plasma membrane of eukaryotic cells where it triggers the internalization of large vacuoles by macropinocytosis. Then, Vice stabilizes these compartments by perturbing the ESCRT machinery. Collectively, our results reveal that Vice is an essential C. burnetii effector protein capable of hijacking two major cellular pathways to shape the bacterial replicative niche.

Vector competence of the African argasid tick Ornithodoros moubata for the Q fever agent Coxiella burnetii.

Q fever is a widespread zoonotic disease caused by the intracellular bacterium Coxiella burnetii. While transmission is primarily but not exclusively airborne, ticks are usually thought to act as vectors on the basis of early microscopy studies. However, recent observations revealed that endosymbionts of ticks have been commonly misidentified as C. burnetii, calling the importance of tick-borne transmission into question. In this study, we re-evaluated the vector competence of the African soft tick Ornithodoros moubata for an avirulent strain of C. burnetii. To this end, we used an artificial feeding system to initiate infection of ticks, specific molecular tools to monitor further infections, and culture assays in axenic and cell media to check for the viability of C. burnetii excreted by ticks. We observed typical traits associated with vector competence: The exposure to an infected blood meal resulted in viable and persistent infections in ticks, trans-stadial transmissions of infection from nymphs to adults and the ability of adult ticks to transmit infectious C. burnetii. However, in contrast to early studies, we found that infection differed substantially between tick organs. In addition, while adult female ticks were infected, we did not observe C. burnetii in eggs, suggesting that transovarial transmission is not effective. Finally, we detected only a sporadic presence of C. burnetii DNA in tick faeces, but no living bacterium was further isolated in culture assays, suggesting that excretion in faeces is not a common mode of transmission in O. moubata.

Coxiella effector protein CvpF subverts RAB26-dependent autophagy to promote vacuole biogenesis and virulence.

Coxiella burnetii, the etiological agent of the zoonosis Q fever, replicates inside host cells within a large vacuole displaying autolysosomal characteristics. The development of this compartment is mediated by bacterial effectors, which interfere with a number of host membrane trafficking pathways. By screening a Coxiella transposon mutant library, we observed that transposon insertions in cbu0626 led to intracellular replication and vacuole biogenesis defects. Here, we demonstrate that CBU0626 is a novel member of the Coxiella vacuolar protein (Cvp) family of effector proteins, which is translocated by the Dot/Icm secretion system and localizes to vesicles with autolysosomal features as well as Coxiella-containing vacuoles (CCVs). We thus renamed this effector CvpF for Coxiella vacuolar protein F. CvpF specifically interacts with the host small GTPase RAB26, leading to the recruitment of the autophagosomal marker MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) to CCVs. Importantly, cvpF::Tn mutants were highly attenuated compared to wild-type bacteria in the SCID mouse model of infection, highlighting the importance of CvpF for Coxiella virulence. These results suggest that CvpF manipulates endosomal trafficking and macroautophagy/autophagy induction for optimal C. burnetii vacuole biogenesis.Abbreviations: ACCM: acidified citrate cystein medium; AP: adaptor related protein complex; CCV: Coxiella-containing vacuole; Cvp: Coxiella vacuolar protein; GDI: guanosine nucleotide dissociation inhibitor; GDF: GDI dissociation factor; GEF: guanine exchange factor; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase MTOR complex 1; PBS: phosphate-buffered saline; PMA: phorbol myristate acetate; SQSTM1/p62: sequestosome 1; WT: wild-type.

The Coxiella burnetii T4SS Effector AnkF Is Important for Intracellular Replication.

Coxiella burnetii is an obligate intracellular pathogen and the causative agent of the zoonotic disease Q fever. Following uptake by alveolar macrophages, the pathogen replicates in an acidic phagolysosomal vacuole, the C. burnetii-containing vacuole (CCV). Effector proteins translocated into the host cell by the type IV secretion system (T4SS) are important for the establishment of the CCV. Here we focus on the effector protein AnkF and its role in establishing the CCV. The C. burnetii AnkF knock out mutant invades host cells as efficiently as wild-type C. burnetii, but this mutant is hampered in its ability to replicate intracellularly, indicating that AnkF might be involved in the development of a replicative CCV. To unravel the underlying reason(s), we searched for AnkF interactors in host cells and identified vimentin through a yeast two-hybrid approach. While AnkF does not alter vimentin expression at the mRNA or protein levels, the presence of AnkF results in structural reorganization and vesicular co-localization with recombinant vimentin. Ectopically expressed AnkF partially accumulates around the established CCV and endogenous vimentin is recruited to the CCV in a time-dependent manner, suggesting that AnkF might attract vimentin to the CCV. However, knocking-down endogenous vimentin does not affect intracellular replication of C. burnetii. Other cytoskeletal components are recruited to the CCV and might compensate for the lack of vimentin. Taken together, AnkF is essential for the establishment of the replicative CCV, however, its mode of action is still elusive.

The secreted protein kinase CstK from Coxiella burnetii influences vacuole development and interacts with the GTPase-activating host protein TBC1D5.

The intracellular bacterial pathogen Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to its pathogenesis is type 4b secretion system-mediated secretion of bacterial effectors into host cells that subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole for intracellular replication. The characterization of prokaryotic serine/threonine protein kinases in bacterial pathogens is emerging as an important strategy to better understand host-pathogen interactions. In this study, we investigated CstK (for Coxiella Ser/Thr kinase), a protein kinase identified in C. burnetii by in silico analysis. We demonstrate that this putative protein kinase undergoes autophosphorylation on Thr and Tyr residues and phosphorylates a classical eukaryotic protein kinase substrate in vitro This dual Thr-Tyr kinase activity is also observed for a eukaryotic dual-specificity Tyr phosphorylation-regulated kinase class. We found that CstK is translocated during infections and localizes to Coxiella-containing vacuoles (CCVs). Moreover, a CstK-overexpressing C. burnetii strain displayed a severe CCV development phenotype, suggesting that CstK fine-tunes CCV biogenesis during the infection. Protein-protein interaction experiments identified the Rab7 GTPase-activating protein TBC1D5 as a candidate CstK-specific target, suggesting a role for this host GTPase-activating protein in Coxiella infections. Indeed, CstK co-localized with TBC1D5 in noninfected cells, and TBC1D5 was recruited to CCVs in infected cells. Accordingly, TBC1D5 depletion from infected cells significantly affected CCV development. Our results indicate that CstK functions as a bacterial effector protein that interacts with the host protein TBC1D5 during vacuole biogenesis and intracellular replication.

Multiple Substrate Usage of Coxiella burnetii to Feed a Bipartite Metabolic Network.

The human pathogen Coxiella burnetii causes Q-fever and is classified as a category B bio-weapon. Exploiting the development of the axenic growth medium ACCM-2, we have now used 13C-labeling experiments and isotopolog profiling to investigate the highly diverse metabolic network of C. burnetii. To this aim, C. burnetii RSA 439 NMII was cultured in ACCM-2 containing 5 mM of either [U-13C3]serine, [U-13C6]glucose, or [U-13C3]glycerol until the late-logarithmic phase. GC/MS-based isotopolog profiling of protein-derived amino acids, methanol-soluble polar metabolites, fatty acids, and cell wall components (e.g., diaminopimelate and sugars) from the labeled bacteria revealed differential incorporation rates and isotopolog profiles. These data served to decipher the diverse usages of the labeled substrates and the relative carbon fluxes into the core metabolism of the pathogen. Whereas, de novo biosynthesis from any of these substrates could not be found for histidine, isoleucine, leucine, lysine, phenylalanine, proline and valine, the other amino acids and metabolites under study acquired 13C-label at specific rates depending on the nature of the tracer compound. Glucose was directly used for cell wall biosynthesis, but was also converted into pyruvate (and its downstream metabolites) through the glycolytic pathway or into erythrose 4-phosphate (e.g., for the biosynthesis of tyrosine) via the non-oxidative pentose phosphate pathway. Glycerol efficiently served as a gluconeogenetic substrate and could also be used via phosphoenolpyruvate and diaminopimelate as a major carbon source for cell wall biosynthesis. In contrast, exogenous serine was mainly utilized in downstream metabolic processes, e.g., via acetyl-CoA in a complete citrate cycle with fluxes in the oxidative direction and as a carbon feed for fatty acid biosynthesis. In summary, the data reflect multiple and differential substrate usages by C. burnetii in a bipartite-type metabolic network, resembling the overall topology of the related pathogen Legionella pneumophila. These strategies could benefit the metabolic capacities of the pathogens also as a trait to adapt for replication under intracellular conditions.

Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development.

The Q fever bacterium Coxiella burnetii replicates inside host cells within a large Coxiella-containing vacuole (CCV) whose biogenesis relies on the Dot/Icm-dependent secretion of bacterial effectors. Several membrane trafficking pathways contribute membranes, proteins, and lipids for CCV biogenesis. These include the endocytic and autophagy pathways, which are characterized by phosphatidylinositol 3-phosphate [PI(3)P]-positive membranes. Here we show that the C. burnetii secreted effector Coxiella vacuolar protein B (CvpB) binds PI(3)P and phosphatidylserine (PS) on CCVs and early endosomal compartments and perturbs the activity of the phosphatidylinositol 5-kinase PIKfyve to manipulate PI(3)P metabolism. CvpB association to early endosome triggers vacuolation and clustering, leading to the channeling of large PI(3)P-positive membranes to CCVs for vacuole expansion. At CCVs, CvpB binding to early endosome- and autophagy-derived PI(3)P and the concomitant inhibition of PIKfyve favor the association of the autophagosomal machinery to CCVs for optimal homotypic fusion of the Coxiella-containing compartments. The importance of manipulating PI(3)P metabolism is highlighted by mutations in cvpB resulting in a multivacuolar phenotype, rescuable by gene complementation, indicative of a defect in CCV biogenesis. Using the insect model Galleria mellonella, we demonstrate the in vivo relevance of defective CCV biogenesis by highlighting an attenuated virulence phenotype associated with cvpB mutations.

Generation and multi-phenotypic high-content screening of Coxiella burnetii transposon mutants.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Identification of OmpA, a Coxiella burnetii protein involved in host cell invasion, by multi-phenotypic high-content screening.

Coxiella burnetii is the agent of the emerging zoonosis Q fever. This pathogen invades phagocytic and non-phagocytic cells and uses a Dot/Icm secretion system to co-opt the endocytic pathway for the biogenesis of an acidic parasitophorous vacuole where Coxiella replicates in large numbers. The study of the cell biology of Coxiella infections has been severely hampered by the obligate intracellular nature of this microbe, and Coxiella factors involved in host/pathogen interactions remain to date largely uncharacterized. Here we focus on the large-scale identification of Coxiella virulence determinants using transposon mutagenesis coupled to high-content multi-phenotypic screening. We have isolated over 3000 Coxiella mutants, 1082 of which have been sequenced, annotated and screened. We have identified bacterial factors that regulate key steps of Coxiella infections: 1) internalization within host cells, 2) vacuole biogenesis/intracellular replication, and 3) protection of infected cells from apoptosis. Among these, we have investigated the role of Dot/Icm core proteins, determined the role of candidate Coxiella Dot/Icm substrates previously identified in silico and identified additional factors that play a relevant role in Coxiella pathogenesis. Importantly, we have identified CBU_1260 (OmpA) as the first Coxiella invasin. Mutations in ompA strongly decreased Coxiella internalization and replication within host cells; OmpA-coated beads adhered to and were internalized by non-phagocytic cells and the ectopic expression of OmpA in E. coli triggered its internalization within cells. Importantly, Coxiella internalization was efficiently inhibited by pretreating host cells with purified OmpA or by incubating Coxiella with a specific anti-OmpA antibody prior to host cell infection, suggesting the presence of a cognate receptor at the surface of host cells. In summary, we have developed multi-phenotypic assays for the study of host/pathogen interactions. By applying our methods to Coxiella burnetii, we have identified the first Coxiella protein involved in host cell invasion.

Quantification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae in French Mediterranean coastal lagoons.

Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae are human pathogens. Little is known about these Vibrio spp. in the coastal lagoons of France. The purpose of this study was to investigate their incidence in water, shellfish and sediment of three French Mediterranean coastal lagoons using the most probable number-polymerase chain reaction (MPN-PCR). In summer, the total number of V. parahaemolyticus in water, sediment, mussels and clams collected from the three lagoons varied from 1 to >1.1 × 10³ MPN/l, 0.09 to 1.1 × 10³ MPN/ml, 9 to 210 MPN/g and 1.5 to 2.1 MPN/g, respectively. In winter, all samples except mussels contained V. parahaemolyticus, but at very low concentrations. Pathogenic (tdh- or trh2-positive) V. parahaemolyticus were present in water, sediment and shellfish samples collected from these lagoons. The number of V. vulnificus in water, sediment and shellfish samples ranged from 1 to 1.1 × 10³ MPN/l, 0.07 to 110 MPN/ml and 0.04 to 15 MPN/g, respectively, during summer. V. vulnificus was not detected during winter. V. cholerae was rarely detected in water and sediment during summer. In summary, results of this study highlight the finding that the three human pathogenic Vibrio spp. are present in the lagoons and constitute a potential public health hazard.

Individual variability of mytimycin gene expression in mussel.

The antifungal peptide mytimycin (MytM) is synthesized by hemocytes of the Mediterranean mussel, Mytilus galloprovincialis. In addition to sequence and gene structure diversities previously reported from pooled hemocytes, the present report focused on the expression of mytm gene in individual M. galloprovincialis, before and after challenge. Within untreated mussel, MytM mRNA was observed by ISH in about 42% of circulating hemocytes, characterized by large, diffuse nucleus. Injection with Fusarium oxysporum increased such percentage, but in only some of the mussels. Similarly, MytM gene expression increased after injection in only some of the mussels, as measured by qPCR. Responders and not responders are common evidence in any given population of organisms. Nevertheless, even if the use of proper pool size selection has been practised to find out and evaluate the most common response trends, individual analyses must be regarded as optimal.

Gene expression specificity of the mussel antifungal mytimycin (MytM).

We previously reported the nucleotide sequences and diversity of mytimycin (MytM) from the Mediterranean mussel, Mytilus galloprovincialis. Using real-time PCR (q-PCR), we observed that the MytM gene was mainly expressed in circulating hemocytes and to a less extent in the mantle. In vivo challenge with bacteria or with the yeast, Candida albicans, did not increase the expression as measured by q-PCR in hemocytes. By contrast, injection of the filamentous fungus, Fusarium oxysporum, induced a sudden and strong increase of expression at 9h p.i. (stimulation index of 25.7 ± 2.1). Optimum stimulating dose was 10(4) spores of F. oxysporum per mussel. In the same samples, AMP mytilin and myticin showed no stimulation. Consequently, we hypothesized the existence of 2 different signal transduction pathways, one activated by bacteria and yeast, the other triggered by filamentous fungi. A second challenge performed with F. oxysporum 24 h after the first challenge induced an increase of MytM gene expression (stimulation index of 3.5 ± 1.7). However, this second increase was significantly lower than the first, suggesting less efficient response rather than significant protection.

Cultivation-independent characterization of methylobacterium populations in the plant phyllosphere by automated ribosomal intergenic spacer analysis.

Bacteria of the genus Methylobacterium are widespread in the environment, but their ecological role in ecosystems, such as the plant phyllosphere, is not very well understood. To gain better insight into the distribution of different Methylobacterium species in diverse ecosystems, a rapid and specific cultivation-independent method for detection of these organisms and analysis of their community structure is needed. Therefore, 16S rRNA gene-targeted primers specific for this genus were designed and evaluated. These primers were used in PCR in combination with a reverse primer that binds to the tRNA(Ala) gene, which is located upstream of the 23S rRNA gene in the 16S-23S intergenic spacer (IGS). PCR products that were of different lengths were obtained due to the length heterogeneity of the IGS of different Methylobacterium species. This length variation allowed generation of fingerprints of Methylobacterium communities in environmental samples by automated ribosomal intergenic spacer analysis. The Methylobacterium communities on leaves of different plant species in a natural field were compared using this method. The new method allows rapid comparisons of Methylobacterium communities and is thus a useful tool to study Methylobacterium communities in different ecosystems.

A plasmid-borne truncated luxI homolog controls quorum-sensing systems and extracellular carbohydrate production in Methylobacterium extorquens AM1.

A cryptic plasmid of Methylobacterium extorquens AM1 was found to encode tslI, a truncated luxI homolog. tslI was shown to be expressed and to control transcription of the acyl-homoserine lactone (HSL) synthase gene msaI and thus, indirectly, acyl-HSL production. In addition, tslI was found to positively regulate extracellular polysaccharide production.

Methylobacterium extorquens AM1 produces a novel type of acyl-homoserine lactone with a double unsaturated side chain under methylotrophic growth conditions.

Acyl-homoserine lactones (acyl-HSLs) have emerged as important regulatory molecules for many gram-negative bacteria. We have found that Methylobacterium extorquens AM1, a member of the pink-pigmented facultative methylotrophs commonly present on plant surfaces, produces several acyl-HSLs depending upon the carbon source. A novel HSL was discovered with a double unsaturated carbon chain (N-(tetradecenoyl)) (C14:2) and characterized by MS and proton NMR. This long-chain acyl-HSL is synthesized by MlaI that also directs synthesis of C14:1-HSL. The Alphaproteobacterium also produces N-hexanoyl-HSL (C6-HSL) and N-octanoyl-HSL (C8-HSL) via MsaI.

Characterization of the genes rdxA and frxA involved in metronidazole resistance in Helicobacter pylori.

Metronidazole (Mtz) resistance in Helicobacter pylori has been found to be associated with mutations in rdxA, a gene encoding an oxygen-insensitive NADPH nitroreductase, and enhanced by mutations in frxA, a gene encoding a NAD(P)H-flavin oxidoreductase. The roles of these two genes in Mtz resistance in H. pylori were examined in this study. The rdxA and frxA genes were sequenced in nine pairs of strains isolated from biopsies obtained from patients before and after failed eradication treatments which included Mtz and resulted in the appearance of resistant strains. Metronidazole resistance could be explained in seven of these pairs of strains by mutations in rdxA and frxA. However, in one pair of strains, rdxA was identical in the susceptible and resistant strains, and only changes in frxA were observed; and in another pair, neither rdxA nor frxA were different in the susceptible and resistant strains. Sequencing of the upstream region of frxA and of the recA gene in the latter pair of strains did not reveal any mutations. To establish whether mutations in frxA alone could be involved in Mtz resistance, a resistant Escherichia coli strain transformed with the frxA of a Mtz susceptible H. pylori strain was rendered susceptible, and transformation with a mutated H. pylori frxA gene under the same conditions did not change the resistant E. coli phenotype. The results suggested that a Mtz resistance phenotype may arise in H. pylori without mutations in rdxA or frxA, or with mutations only in frxA.

Which test to use to detect Helicobacter pylori infection in patients with low-grade gastric mucosa-associated lymphoid tissue lymphoma?

Helicobacter pylori is involved in the pathogenesis of lymphoma of the gastric mucosa-associated lymphoid tissue (MALT). Because gastric MALT lymphoma is a rare disease, few studies comparing the accuracy of diagnostic tests in this group of patients have been carried out, and only a limited number of tests (essentially histological) were performed. The aim of our study was to compare the results of four different diagnostic methods used to detect H. pylori (histology, culture, polymerase chain reaction, and serology) in a prospective multicenter study. A patient was considered to be H. pylori positive if culture or histology was positive. During the period 1995-2000, a total of 90 patients with low-grade gastric MALT lymphoma were enrolled. Results for the four tests were available for 56 patients (62.2%). Among these patients, the four tests were concordant in 35 cases (62.5%), i.e., were positive in 19 cases (33.9%) and negative in 16 patients (17.8%). Histology (39/40 positive, 97.5%) and serology (38/40 positive, 95.0%) were the most sensitive tests. Polymerase chain reaction (PCR) and culture were positive in 52.5% and 50%, respectively. The cagA gene was detected in 47.4% of the strains.

Discriminatory power of RAPD, PCR-RFLP and southern blot analyses of ureCD or ureA gene probes on Helicobacter pylori isolates.

The genetic diversity of 33 Nigerian Helicobacter pylori isolates were studied using RAPD, PCR-RFLP and Southern blot analysis of ureA or ureCD gene probes. RAPD was able to distinguish the following number of isolates using the primers 3880: 5'-AAGAGCCCGT-3' (28), 3881 :5'-AACGCGCAAC-3' (33) and OPH8 :5'-GAAACACCCC-3' (25). Southern blot analysis using the ureCD probe was also able to distinguish the 12 isolates tested into ten different patterns. The PCR-RFLP technique distinguished all 33 isolates into six types. In conclusion, considering typeability, discriminatory power, and convenience, RAPD with the 3881 primer was considered the most useful technique.