FLO1 is a variable green beard gene that drives biofilm-like cooperation in budding yeast.

The budding yeast, Saccharomyces cerevisiae, has emerged as an archetype of eukaryotic cell biology. Here we show that S. cerevisiae is also a model for the evolution of cooperative behavior by revisiting flocculation, a self-adherence phenotype lacking in most laboratory strains. Expression of the gene FLO1 in the laboratory strain S288C restores flocculation, an altered physiological state, reminiscent of bacterial biofilms. Flocculation protects the FLO1 expressing cells from multiple stresses, including antimicrobials and ethanol. Furthermore, FLO1(+) cells avoid exploitation by nonexpressing flo1 cells by self/non-self recognition: FLO1(+) cells preferentially stick to one another, regardless of genetic relatedness across the rest of the genome. Flocculation, therefore, is driven by one of a few known "green beard genes," which direct cooperation toward other carriers of the same gene. Moreover, FLO1 is highly variable among strains both in expression and in sequence, suggesting that flocculation in S. cerevisiae is a dynamic, rapidly evolving social trait.

Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells.

Listeria monocytogenes causes listeriosis, a foodborne disease that poses serious risks to fetuses, newborns and immunocompromised adults. This intracellular bacterial pathogen proliferates in the host cytosol and exploits the host actin polymerization machinery to spread from cell-to-cell and disseminate in the host. Here, we report that during several days of infection in human hepatocytes or trophoblast cells, L. monocytogenes switches from this active motile lifestyle to a stage of persistence in vacuoles. Upon intercellular spread, bacteria gradually stopped producing the actin-nucleating protein ActA and became trapped in lysosome-like vacuoles termed Listeria-Containing Vacuoles (LisCVs). Subpopulations of bacteria resisted degradation in LisCVs and entered a slow/non-replicative state. During the subculture of host cells harboring LisCVs, bacteria showed a capacity to cycle between the vacuolar and the actin-based motility stages. When ActA was absent, such as in ΔactA mutants, vacuolar bacteria parasitized host cells in the so-called "viable but non-culturable" state (VBNC), preventing their detection by conventional colony counting methods. The exposure of infected cells to high doses of gentamicin did not trigger the formation of LisCVs, but selected for vacuolar and VBNC bacteria. Together, these results reveal the ability of L. monocytogenes to enter a persistent state in a subset of epithelial cells, which may favor the asymptomatic carriage of this pathogen, lengthen the incubation period of listeriosis, and promote bacterial survival during antibiotic therapy.

MybA, a transcription factor involved in conidiation and conidial viability of the human pathogen Aspergillus fumigatus.

Aspergillus fumigatus, a ubiquitous human fungal pathogen, produces asexual spores (conidia), which are the main mode of propagation, survival and infection of this human pathogen. In this study, we present the molecular characterization of a novel regulator of conidiogenesis and conidial survival called MybA because the predicted protein contains a Myb DNA binding motif. Cellular localization of the MybA::Gfp fusion and immunoprecipitation of the MybA::Gfp or MybA::3xHa protein showed that MybA is localized to the nucleus. RNA sequencing data and a uidA reporter assay indicated that the MybA protein functions upstream of wetA, vosA and velB, the key regulators involved in conidial maturation. The deletion of mybA resulted in a very significant reduction in the number and viability of conidia. As a consequence, the ΔmybA strain has a reduced virulence in an experimental murine model of aspergillosis. RNA-sequencing and biochemical studies of the ΔmybA strain suggested that MybA protein controls the expression of enzymes involved in trehalose biosynthesis as well as other cell wall and membrane-associated proteins and ROS scavenging enzymes. In summary, MybA protein is a new key regulator of conidiogenesis and conidial maturation and survival, and plays a crucial role in propagation and virulence of A. fumigatus.

A Large Collection of Novel Nematode-Infecting Microsporidia and Their Diverse Interactions with Caenorhabditis elegans and Other Related Nematodes.

Microsporidia are fungi-related intracellular pathogens that may infect virtually all animals, but are poorly understood. The nematode Caenorhabditis elegans has recently become a model host for studying microsporidia through the identification of its natural microsporidian pathogen Nematocida parisii. However, it was unclear how widespread and diverse microsporidia infections are in C. elegans or other related nematodes in the wild. Here we describe the isolation and culture of 47 nematodes with microsporidian infections. N. parisii is found to be the most common microsporidia infecting C. elegans in the wild. In addition, we further describe and name six new species in the Nematocida genus. Our sampling and phylogenetic analysis further identify two subclades that are genetically distinct from Nematocida, and we name them Enteropsectra and Pancytospora. Interestingly, unlike Nematocida, these two genera belong to the main clade of microsporidia that includes human pathogens. All of these microsporidia are horizontally transmitted and most specifically infect intestinal cells, except Pancytospora epiphaga that replicates mostly in the epidermis of its Caenorhabditis host. At the subcellular level in the infected host cell, spores of the novel genus Enteropsectra show a characteristic apical distribution and exit via budding off of the plasma membrane, instead of exiting via exocytosis as spores of Nematocida. Host specificity is broad for some microsporidia, narrow for others: indeed, some microsporidia can infect Oscheius tipulae but not its sister species Oscheius sp. 3, and conversely some microsporidia found infecting Oscheius sp. 3 do not infect O. tipulae. We also show that N. ausubeli fails to strongly induce in C. elegans the transcription of genes that are induced by other Nematocida species, suggesting it has evolved mechanisms to prevent induction of this host response. Altogether, these newly isolated species illustrate the diversity and ubiquity of microsporidian infections in nematodes, and provide a rich resource to investigate host-parasite coevolution in tractable nematode hosts.

Anoxia and glucose supplementation preserve neutrophil viability and function.

Functional studies of human neutrophils and their transfusion for clinical purposes have been hampered by their short life span after isolation. Here, we demonstrate that neutrophil viability is maintained for 20 hours in culture media at 37°C under anoxic conditions with 3 mM glucose and 32 µg/mL dimethyloxalylglycine supplementation, as evidenced by stabilization of Mcl-1, proliferating cell nuclear antigen (PCNA), and pro-caspase-3. Notably, neutrophil morphology (nucleus shape and cell-surface markers) and functions (phagocytosis, degranulation, calcium release, chemotaxis, and reactive oxygen species production) were comparable to blood circulating neutrophils. The observed extension in neutrophil viability was reversed upon exposure to oxygen. Extending neutrophil life span allowed efficient transfection of plasmids (40% transfection efficiency) and short interfering RNA (interleukin-8, PCNA, and Bax), as a validation of effective and functional genetic manipulation of neutrophils both in vitro and in vivo. In vivo, transfusion of conditioned neutrophils in a neutropenic guinea pig model increased bacterial clearance of Shigella flexneri upon colonic infection, strongly suggesting that these conditioned neutrophils might be suitable for transfusion purposes. In summary, such conditioning of neutrophils in vitro should facilitate their study and offer new opportunities for genetic manipulation and therapeutic use.

GH16 and GH81 family ß-(1,3)-glucanases in Aspergillus fumigatus are essential for conidial cell wall morphogenesis.

The fungal cell wall is a rigid structure because of fibrillar and branched ß-(1,3)-glucan linked to chitin. Softening of the cell wall is an essential phenomenon during fungal morphogenesis, wherein rigid cell wall structures are cleaved by glycosylhydrolases. During the search for glycosylhydrolases acting on ß-(1,3)-glucan, we identified seven genes in the Aspergillus fumigatus genome coding for potential endo-ß-(1,3)-glucanase. ENG1 (previously characterized and named ENGL1, Mouyna et al., ), belongs to the Glycoside-Hydrolase 81 (GH81) family, while ENG2 to ENG7, to GH16 family. ENG1 and four GH16 genes (ENG2-5) were expressed in the resting conidia as well as during germination, suggesting an essential role during A. fumigatus morphogenesis. Here, we report the effect of sequential deletion of AfENG2-5 (GH16) followed by AfENG1 (GH81) deletion in the Δeng2,3,4,5 mutant. The Δeng1,2,3,4,5 mutant showed conidial defects, with linear chains of conidia unable to separate while the germination rate was not affected. These results show, for the first time in a filamentous fungus, that endo ß-(1,3)-glucanases are essential for proper conidial cell wall assembly and thus segregation of conidia during conidiation.

Biosynthesis of cell wall mannan in the conidium and the mycelium of Aspergillus fumigatus.

The galactomannan is a major cell wall molecule of Aspergillus fumigatus. This molecule is composed of a linear mannan with a repeating unit composed of four α1,6 and α1,2 linked mannose with side chains of galactofuran. To obtain a better understanding of the mannan biosynthesis in A. fumigatus, it was decided to undertake the successive deletion of the 11 genes which are putative orthologs of the mannosyltransferases responsible for establishing α1,6 and α1,2 mannose linkages in yeast. These deletions did not lead to a reduction of the mannan content of the cell wall of the mycelium of A. fumigatus. In contrast, the mannan content of the conidial cell wall was reduced and this reduction was associated with a partial disorganization of the cell wall leading to defects in conidial survival both in vitro and in vivo.

Aspergillus fumigatus devoid of cell wall ß-1,3-glucan is viable, massively sheds galactomannan and is killed by septum formation inhibitors.

Echinocandins inhibit ß-1,3-glucan synthesis and are one of the few antimycotic drug classes effective against Aspergillus spp. In this study, we characterized the ß-1,3-glucan synthase Fks1 of Aspergillus fumigatus, the putative target of echinocandins. Data obtained with a conditional mutant suggest that fks1 is not essential. In agreement, we successfully constructed a viable Δfks1 deletion mutant. Lack of Fks1 results in characteristic growth phenotypes similar to wild type treated with echinocandins and an increased susceptibility to calcofluor white and sodium dodecyl sulfate. In agreement with Fks1 being the only ß-1,3-glucan synthase in A. fumigatus, the cell wall is devoid of ß-1,3-glucan. This is accompanied by a compensatory increase of chitin and galactosaminogalactan and a significant decrease in cell wall galactomannan due to a massively enhanced galactomannan shedding. Our data furthermore suggest that inhibition of hyphal septation can overcome the limitations of echinocandin therapy. Compounds inhibiting septum formation boosted the antifungal activity of caspofungin. Thus, development of clinically applicable inhibitors of septum formation is a promising strategy to improve existing antifungal therapy.

Modulation of Shigella virulence in response to available oxygen in vivo.

Bacteria coordinate expression of virulence determinants in response to localized microenvironments in their hosts. Here we show that Shigella flexneri, which causes dysentery, encounters varying oxygen concentrations in the gastrointestinal tract, which govern activity of its type three secretion system (T3SS). The T3SS is essential for cell invasion and virulence. In anaerobic environments (for example, the gastrointestinal tract lumen), Shigella is primed for invasion and expresses extended T3SS needles while reducing Ipa (invasion plasmid antigen) effector secretion. This is mediated by FNR (fumarate and nitrate reduction), a regulator of anaerobic metabolism that represses transcription of spa32 and spa33, virulence genes that regulate secretion through the T3SS. We demonstrate there is a zone of relative oxygenation adjacent to the gastrointestinal tract mucosa, caused by diffusion from the capillary network at the tips of villi. This would reverse the anaerobic block of Ipa secretion, allowing T3SS activation at its precise site of action, enhancing invasion and virulence.

Deciphering the role of the chitin synthase families 1 and 2 in the in vivo and in vitro growth of Aspergillus fumigatus by multiple gene targeting deletion.

Although chitin is an essential component of the fungal cell wall (CW), its biosynthesis and role in virulence is poorly understood. In Aspergillus fumigatus, there are eight chitin synthase (CHS) genes belonging to two families CHSA-C, CHSG in family 1 and CHSF, CHSD, CSMA, CSMB in family 2). To understand the function of these CHS genes, their single and multiple deletions were performed using ß-rec/six system to be able to delete all genes within each family (up to a quadruple ΔchsA/C/B/G mutant in family 1 and a quadruple ΔcsmA/csmB/F/D mutant in family 2). Radial growth, conidiation, mycelial/conidial morphology, CW polysaccharide content, Chs-activity, susceptibility to antifungal molecules and pathogenicity in experimental animal aspergillosis were analysed for all the mutants. Among the family 1 CHS, ΔchsA, ΔchsB and ΔchsC mutants showed limited impact on chitin synthesis. In contrast, there was reduced conidiation, altered mycelial morphotype and reduced growth and Chs-activity in the ΔchsG and ΔchsA/C/B/G mutants. In spite of this altered phenotype, these two mutants were as virulent as the parental strain in the experimental aspergillosis models. Among family 2 CHS, phenotypic defects mainly resulted from the CSMA deletion. Despite significant morphological mycelial and conidial growth phenotypes in the quadruple ΔcsmA/csmB/F/D mutant, the chitin content was poorly affected by gene deletions in this family. However, the entire mycelial cell wall structure was disorganized in the family 2 mutants that may be related to the reduced pathogenicity of the quadruple ΔcsmA/csmB/F/D mutant strain compared to the parental strain, in vivo. Deletion of the genes encompassing the two families (ΔcsmA/csmB/F/G) showed that in spite of being originated from an ancient divergence of fungi, these two families work cooperatively to synthesize chitin in A. fumigatus and demonstrate the essentiality of chitin biosynthesis for vegetative growth, resistance to antifungal drugs, and virulence of this filamentous fungus.

Species-specific impact of the autophagy machinery on Chikungunya virus infection.

Chikungunya virus (CHIKV) is a recently re-emerged arbovirus that triggers autophagy. Here, we show that CHIKV interacts with components of the autophagy machinery during its replication cycle, inducing a cytoprotective effect. The autophagy receptor p62 protects cells from death by binding ubiquitinated capsid and targeting it to autophagolysosomes. By contrast, the human autophagy receptor NDP52--but not its mouse orthologue--interacts with the non-structural protein nsP2, thereby promoting viral replication. These results highlight the distinct roles of p62 and NDP52 in viral infection, and identify NDP52 as a cellular factor that accounts for CHIKV species specificity.

A natural system of chromosome transfer in Yersinia pseudotuberculosis.

The High Pathogenicity Island of Yersinia pseudotuberculosis IP32637 was previously shown to be horizontally transferable as part of a large chromosomal segment. We demonstrate here that at low temperature other chromosomal loci, as well as a non-mobilizable plasmid (pUC4K), are also transferable. This transfer, designated GDT4 (Generalized DNA Transfer at 4°C), required the presence of an IP32637 endogenous plasmid (pGDT4) that carries several mobile genetic elements and a conjugation machinery. We established that cure of this plasmid or inactivation of its sex pilus fully abrogates this process. Analysis of the mobilized pUC4K recovered from transconjugants revealed the insertion of one of the pGDT4-borne ISs, designated ISYps1, at different sites on the transferred plasmid molecules. This IS belongs to the IS6 family, which moves by replicative transposition, and thus could drive the formation of cointegrates between pGDT4 and the host chromosome and could mediate the transfer of chromosomal regions in an Hfr-like manner. In support of this model, we show that a suicide plasmid carrying ISYps1 is able to integrate itself, flanked by ISYps1 copies, at multiple locations into the Escherichia coli chromosome. Furthermore, we demonstrate the formation of RecA-independent cointegrates between the ISYps1-harboring plasmid and an ISYps1-free replicon, leading to the passive transfer of the non-conjugative plasmid. We thus demonstrate here a natural mechanism of horizontal gene exchange, which is less constrained and more powerful than the classical Hfr mechanism, as it only requires the presence of an IS6-type element on a conjugative replicon to drive the horizontal transfer of any large block of plasmid or chromosomal DNA. This natural mechanism of chromosome transfer, which occurs under conditions mimicking those found in the environment, may thus play a significant role in bacterial evolution, pathogenesis, and adaptation to new ecological niches.

SUN proteins belong to a novel family of ß-(1,3)-glucan-modifying enzymes involved in fungal morphogenesis.

BACKGROUND: SUN proteins are involved in yeast morphogenesis, but their function is unknown. RESULTS: SUN protein plays a role in the Aspergillus fumigatus morphogenesis. Biochemical properties of recombinant SUN proteins were elucidated. CONCLUSION: Both Candida albicans and Aspergillus fumigatus sun proteins show a ß-(1,3)-glucanase activity. SIGNIFICANCE: The mode of action of SUN proteins on ß-(1,3)-glucan is unique, new, and original. In yeasts, the family of SUN proteins has been involved in cell wall biogenesis. Here, we report the characterization of SUN proteins in a filamentous fungus, Aspergillus fumigatus. The function of the two A. fumigatus SUN genes was investigated by combining reverse genetics and biochemistry. During conidial swelling and mycelial growth, the expression of AfSUN1 was strongly induced, whereas the expression of AfSUN2 was not detectable. Deletion of AfSUN1 negatively affected hyphal growth and conidiation. A closer examination of the morphological defects revealed swollen hyphae, leaky tips, intrahyphal growth, and double cell wall, suggesting that, like in yeast, AfSun1p is associated with cell wall biogenesis. In contrast to AfSUN1, deletion of AfSUN2 either in the parental strain or in the AfSUN1 single mutant strain did not affect colony and hyphal morphology. Biochemical characterization of the recombinant AfSun1p and Candida albicans Sun41p showed that both proteins had a unique hydrolysis pattern: acting on ß-(1,3)-oligomers from dimer up to insoluble ß-(1,3)-glucan. Referring to the CAZy database, it is clear that fungal SUN proteins represent a new family of glucan hydrolases (GH132) and play an important morphogenetic role in fungal cell wall biogenesis and septation.

Transcytosis of HTLV-1 across a tight human epithelial barrier and infection of subepithelial dendritic cells.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to blood transfusion and sexual transmission, HTLV-1 is transmitted mainly through prolonged breastfeeding, and such infection represents a major risk for the development of adult T-cell leukemia/lymphoma. Although HTLV-1-infected lymphocytes can be retrieved from maternal milk, the mechanisms of HTLV-1 transmission through the digestive tract remain unknown. In the present study, we assessed HTLV-1 transport across the epithelial barrier using an in vitro model. Our results show that the integrity of the epithelial barrier was maintained during coculture with HTLV-1-infected lymphocytes, because neither morphological nor functional alterations of the cell monolayer were observed. Enterocytes were not susceptible to HTLV-1 infection, but free infectious HTLV-1 virions could cross the epithelial barrier via a transcytosis mechanism. Such virions were able to infect productively human dendritic cells located beneath the epithelial barrier. Our data indicate that HTLV-1 crosses the tight epithelial barrier without disruption or infection of the epithelium to further infect target cells such as dendritic cells. The present study provides the first data pertaining to the mode of HTLV-1 transport across a tight epithelial barrier, as can occur during mother-to-child HTLV-1 transmission during breastfeeding.

Simple and elegant design of a virion egress structure in Archaea.

Some viruses of Archaea use an unusual egress mechanism that involves the formation of virus-associated pyramids (VAPs) on the host cell surface. At the end of the infection cycle, these structures open outward and create apertures through which mature virions escape from the cell. Here we describe in detail the structure and composition of VAPs formed by the Sulfolobus islandicus rod-shaped virus 2 (SIRV2) in cells of its hyperthermophilic archaeal host. We show that the VAPs are stable and autonomous assemblies that can be isolated from membranes of infected cells and purified without affecting their structure. The purified VAPs are heterogeneous in size, reflecting the dynamics of VAP development in a population of infected cells; however, they have a uniform geometry, consisting of seven isosceles triangular faces forming a baseless pyramid. Biochemical and immunoelectron microscopy analyses revealed that the 10-kDa P98 protein encoded by the SIRV2 virus is the sole component of the VAPs. The VAPs were produced in Sulfolobus acidocaldarius and Escherichia coli by heterologous expression of the SIRV2-P98 gene. The results confirm that P98 is the only constituent of the VAPs and demonstrate that no other viral protein is involved in the assembly of pyramids. P98 was able to produce stable structures under conditions ranging from moderate to extremely high temperatures (80 °C) and from neutral to extremely acidic pH (pH 2), demonstrating another remarkable property of this exceptional viral protein.

Peptidoglycan maturation enzymes affect flagellar functionality in bacteria.

The flagellar machinery is a highly complex organelle composed of a free rotating flagellum and a fixed stator that converts energy into movement. The assembly of the flagella and the stator requires interactions with the peptidoglycan layer through which the organelle has to pass for externalization. Lytic transglycosylases are peptidoglycan degrading enzymes that cleave the sugar backbone of peptidoglycan layer. We show that an endogenous lytic transglycosylase is required for full motility of Helicobacter pylori and colonization of the gastric mucosa. Deficiency of motility resulted from a paralysed phenotype implying an altered ability to generate flagellar rotation. Similarly, another Gram-negative pathogen Salmonella typhimurium and the Gram-positive pathogen Listeria monocytogenes required the activity of lytic transglycosylases, Slt or MltC, and a glucosaminidase (Auto), respectively, for full motility. Furthermore, we show that in absence of the appropriate lytic transglycosylase, the flagellar motor protein MotB from H. pylori does not localize properly to the bacterial pole. We present a new model involving the maturation of the surrounding peptidoglycan for the proper anchoring and functionality of the flagellar motor.

Characterization of the elongasome core PBP2 : MreC complex of Helicobacter pylori.

The definition of bacterial cell shape is a complex process requiring the participation of multiple components of an intricate macromolecular machinery. We aimed at characterizing the determinants involved in cell shape of the helical bacterium Helicobacter pylori. Using a yeast two-hybrid screen with the key cell elongation protein PBP2 as bait, we identified an interaction between PBP2 and MreC. The minimal region of MreC required for this interaction ranges from amino acids 116 to 226. Using recombinant proteins, we showed by affinity and size exclusion chromatographies and surface plasmon resonance that PBP2 and MreC form a stable complex. In vivo, the two proteins display a similar spatial localization and their complex has an apparent 1:1 stoichiometry; these results were confirmed in vitro by analytical ultracentrifugation and chemical cross-linking. Small angle X-ray scattering analyses of the PBP2 : MreC complex suggest that MreC interacts directly with the C-terminal region of PBP2. Depletion of either PBP2 or MreC leads to transition into spherical cells that lose viability. Finally, the specific expression in trans of the minimal interacting domain of MreC with PBP2 in the periplasmic space leads to cell rounding, suggesting that the PBP2/MreC complex formation in vivo is essential for cell morphology.

A unique virus release mechanism in the Archaea.

Little is known about the infection cycles of viruses infecting cells from Archaea, the third domain of life. Here, we demonstrate that the virions of the archaeal Sulfolobus islandicus rod-shaped virus 2 (SIRV2) are released from the host cell through a mechanism, involving the formation of specific cellular structures. Large pyramidal virus-induced protrusions transect the cell envelope at several positions, rupturing the S-layer; they eventually open out, thus creating large apertures through which virions escape the cell. We also demonstrate that massive degradation of the host chromosomes occurs because of virus infection, and that virion assembly occurs in the cytoplasm. Furthermore, intracellular viral DNA is visualized by flow cytometry. The results show that SIRV2 is a lytic virus, and that the host cell dies as a consequence of elaborated mechanisms orchestrated by the virus. The generation of specific cellular structures for a distinct step of virus life cycle is known in eukaryal virus-host systems but is unprecedented in cells from other domains.

Regulated shift from helical to polar localization of Listeria monocytogenes cell wall-anchored proteins.

Many virulence factors of Gram-positive bacterial pathogens are covalently anchored to the peptidoglycan (PG) by sortase enzymes. However, for rod-shaped bacteria little is known about the spatiotemporal organization of these surface proteins in the cell wall. Here we report the three-dimensional (3D) localization of the PG-bound virulence factors InlA, InlH, InlJ, and SvpA in the envelope of Listeria monocytogenes under different growth conditions. We found that all PG-anchored proteins are positioned along the lateral cell wall in nonoverlapping helices. However, these surface proteins can also become localized at the pole and asymmetrically distributed when specific regulatory pathways are activated. InlA and InlJ are enriched at poles when expressed at high levels in exponential-phase bacteria. InlA and InlH, which are σ(B)dependent, specifically relocalize to the septal cell wall and subsequently to the new pole in cells entering stationary phase. The accumulation of InlA and InlH in the septal region also occurs when oxidative stress impairs bacterial growth. In contrast, the iron-dependent protein SvpA is present at the old pole and is excluded from the septum and new pole of bacteria grown under low-iron conditions. We conclude that L. monocytogenes rapidly reorganizes the spatial localization of its PG proteins in response to changes in environmental conditions such as nutrient deprivation or other stresses. This dynamic control would distribute virulence factors at specific sites during the infectious process.

A M23B family metallopeptidase of Helicobacter pylori required for cell shape, pole formation and virulence.

The molecular basis of the regulation of specific shapes and their role for the bacterial fitness remain largely unknown. We focused in this study on the Gram-negative and spiral-shaped Helicobacter pylori. To colonize its unique niche, H. pylori needs to reach quickly the human gastric mucosa, by swimming to and through the mucus layer. For that reason, the specific shape of H. pylori is predicted to be necessary for optimal motility in vivo, and consequently for its colonization ability. Here, we describe the involvement of a PG-modifying enzyme, HdpA (HP0506), in the mouse colonization ability of this bacterium, by regulating its shape. Indeed, the inactivation of the hp0506 gene led to a stocky and branched phenotype, affecting H. pylori colonization capacity despite a normal motility phenotype in vitro. In contrast, the overexpression of the hp0506 gene induced the transformation of H. pylori from rod to dividing cocci shaped bacteria. Furthermore, we demonstrated by PG analysis and enzymology, that HdpA carried both d,d-carboxypeptidase and d,d-endopeptidase activities. Thus, HdpA is the first enzyme belonging to the M23-peptidase family able to perform the d,d-carboxypeptidation and regulate cell shape.