Peptidoglycan crosslinking relaxation promotes Helicobacter pylori's helical shape and stomach colonization.

The mechanisms by which bacterial cells generate helical cell shape and its functional role are poorly understood. Helical shape of the human pathogen Helicobacter pylori may facilitate penetration of the thick gastric mucus where it replicates. We identified four genes required for helical shape: three LytM peptidoglycan endopeptidase homologs (csd1-3) and a ccmA homolog. Surrounding the cytoplasmic membrane of most bacteria, the peptidoglycan (murein) sacculus is a meshwork of glycan strands joined by peptide crosslinks. Intact cells and isolated sacculi from mutants lacking any single csd gene or ccmA formed curved rods and showed increased peptidoglycan crosslinking. Quantitative morphological analyses of multiple-gene deletion mutants revealed each protein uniquely contributes to a shape-generating pathway. This pathway is required for robust colonization of the stomach in spite of normal directional motility. Our findings suggest that the coordinated action of multiple proteins relaxes peptidoglycan crosslinking, enabling helical cell curvature and twist.

A peptide of a type I toxin-antitoxin system induces Helicobacter pylori morphological transformation from spiral shape to coccoids.

Toxin-antitoxin systems are found in many bacterial chromosomes and plasmids with roles ranging from plasmid stabilization to biofilm formation and persistence. In these systems, the expression/activity of the toxin is counteracted by an antitoxin, which, in type I systems, is an antisense RNA. While the regulatory mechanisms of these systems are mostly well defined, the toxins' biological activity and expression conditions are less understood. Here, these questions were investigated for a type I toxin-antitoxin system (AapA1-IsoA1) expressed from the chromosome of the human pathogen Helicobacter pylori We show that expression of the AapA1 toxin in H. pylori causes growth arrest associated with rapid morphological transformation from spiral-shaped bacteria to round coccoid cells. Coccoids are observed in patients and during in vitro growth as a response to different stress conditions. The AapA1 toxin, first molecular effector of coccoids to be identified, targets H. pylori inner membrane without disrupting it, as visualized by cryoelectron microscopy. The peptidoglycan composition of coccoids is modified with respect to spiral bacteria. No major changes in membrane potential or adenosine 5'-triphosphate (ATP) concentration result from AapA1 expression, suggesting coccoid viability. Single-cell live microscopy tracking the shape conversion suggests a possible association of this process with cell elongation/division interference. Oxidative stress induces coccoid formation and is associated with repression of the antitoxin promoter and enhanced processing of its transcript, leading to an imbalance in favor of AapA1 toxin expression. Our data support the hypothesis of viable coccoids with characteristics of dormant bacteria that might be important in H. pylori infections refractory to treatment.

Colonization of Helicobacter pylori in the oral cavity - an endless controversy?

Helicobacter pylori is associated with chronic gastritis, gastric or duodenal ulcers, and gastric cancer. Since the oral cavity is the entry port and the first component of the gastrointestinal system, the oral cavity has been discussed as a potential reservoir of H. pylori. Accordingly, a potential oral-oral transmission route of H. pylori raises the question concerning whether close contact such as kissing or sharing a meal can cause the transmission of H. pylori. Therefore, this topic has been investigated in many studies, applying different techniques for detection of H. pylori from oral samples, i.e. molecular techniques, immunological or biochemical methods and traditional culture techniques. While molecular, immunological or biochemical methods usually yield high detection rates, there is no definitive evidence that H. pylori has ever been isolated from the oral cavity. The specificity of those methods may be limited due to potential cross-reactivity, especially with H. pylori-like microorganisms such as Campylobacter spp. Furthermore, the influence of gastroesophageal reflux has not been investigated so far. This review aims to summarize and critically discuss previous studies investigating the potential colonization of H. pylori in the oral cavity and suggest novel research directions for targeting this critical research question.

Mucoid and coccoid Helicobacter pylori with fast growth and antibiotic resistance.

BACKGROUND: In this study, one Helicobacter pylori isolate, from gastric biopsy of a dyspeptic patient that turned into mucoid-coccoid (MC) form upon consecutive subcultures, was identified. The culturability, antibiotic resistance, and lipid contents of MC were compared with those of non-mucoid (NM) spiral H pylori. MATERIALS AND METHODS: Mucoid-coccoid and NM H pylori were subcultured on Brucella blood agar (BBA) and incubated under aerobic and microaerobic atmospheres at 37°C. Cultures were examined for colony characteristics and bacterial morphology after 1-3 days. The isolates were identified by biochemical tests and detection of H pylori-16S rDNA. Antibiogram was performed with currently used antibiotics for H pylori eradication. Cellular lipid contents were extracted and analyzed by gas chromatography. RESULTS: Compared with pin-pointed and glistening colonies of NM H pylori that appeared under microaerobic conditions, MC H pylori grew well in consecutive subcultures under aerobic and microaerobic atmospheres and produced white patches of mucoid colonies. MC exhibited coccoid and NM spiral morphology. Both isolates were catalase, oxidase, and urease positive and contained 16S rDNA. Compared with NM that was susceptible to almost all the antibiotics, MC was resistant to all the antibiotics. Lipid analyses showed high frequency of unsaturated fatty acids and cholesterol in MC. CONCLUSIONS: Coccoid forms with high fatty acid and cholesterol contents that show resistance to antibiotics might resist against other stressful conditions such as gastric acidity and immune response. Moreover, mucoid property may enhance resistance of coccoids to stresses. With mucoid-coccoid lifestyle, H pylori may establish a chronic infection refractory to antimicrobial therapy.

N-monoarylacetothioureas as potent urease inhibitors: synthesis, SAR, and biological evaluation.

A urease inhibitor with good in vivo profile is considered as an alternative agent for treating infections caused by urease-producing bacteria such as Helicobacter pylori. Here, we report a series of N-monosubstituted thioureas, which act as effective urease inhibitors with very low cytotoxicity. One compound (b19) was evaluated in detail and shows promising features for further development as an agent to treat H. pylori caused diseases. Excellent values for the inhibition of b19 against both extracted urease and urease in intact cell were observed, which shows IC50 values of 0.16 ± 0.05 and 3.86 ± 0.10 µM, being 170- and 44-fold more potent than the clinically used drug AHA, respectively. Docking simulations suggested that the monosubstituted thiourea moiety penetrates urea binding site. In addition, b19 is a rapid and reversible urease inhibitor, and displays nM affinity to urease with very slow dissociation (koff=1.60 × 10-3 s-1) from the catalytic domain.

A Genome-Wide Helicobacter pylori Morphology Screen Uncovers a Membrane-Spanning Helical Cell Shape Complex.

Evident in its name, the gastric pathogen Helicobacter pylori has a helical cell morphology which facilitates efficient colonization of the human stomach. An improved light-focusing strategy allowed us to robustly distinguish even subtle perturbations of H. pylori cell morphology by deviations in light-scattering properties measured by flow cytometry. Profiling of an arrayed genome-wide deletion library identified 28 genes that influence different aspects of cell shape, including properties of the helix, cell length or width, cell filament formation, cell shape heterogeneity, and cell branching. Included in this mutant collection were two that failed to form any helical cells, a soluble lytic transglycosylase and a previously uncharacterized putative multipass inner membrane protein HPG27_0728, renamed Csd7. A combination of cell fractionation, mutational, and immunoprecipitation experiments show that Csd7 and Csd2 collaborate to stabilize the Csd1 peptidoglycan (PG) endopeptidase. Thus, both csd2 and csd7 mutants show the same enhancement of PG tetra-pentapeptide cross-linking as csd1 mutants. Csd7 also links Csd1 with the bactofilin CcmA via protein-protein interactions. Although Csd1 is stable in ccmA mutants, these mutants show altered PG tetra-pentapeptide cross-linking, suggesting that Csd7 may directly or indirectly activate as well as stabilize Csd1. These data begin to illuminate a highly orchestrated program to regulate PG modifications that promote helical shape, which includes nine nonessential nonredundant genes required for helical shape and 26 additional genes that further modify H. pylori's cell morphology.IMPORTANCE The stomach ulcer and cancer-causing pathogen Helicobacter pylori has a helical cell shape which facilitates stomach infection. Using light scattering to measure perturbations of cell morphology, we identified 28 genes that influence different aspects of cell shape. A mutant in a previously uncharacterized protein renamed Csd7 failed to form any helical cells. Biochemical analyses showed that Csd7 collaborates with other proteins to stabilize the cell wall-degrading enzyme Csd1. Csd7 also links Csd1 with a putative filament-forming protein via protein-protein interactions. These data suggest that helical cell shape arises from a highly orchestrated program to regulate cell wall modifications. Targeting of this helical cell shape-promoting program could offer new ways to block infectivity of this important human pathogen.

Nonhelical Helicobacter pylori Mutants Show Altered Gland Colonization and Elicit Less Gastric Pathology than Helical Bacteria during Chronic Infection.

Half of all humans harbor Helicobacter pylori in their stomachs. Helical cell shape is thought to facilitate H. pylori's ability to bore into the protective mucus layer in a corkscrew-like motion, thereby enhancing colonization of the stomach. H. pylori cell shape mutants show impaired colonization of the mouse stomach, highlighting the importance of cell shape in infection. To gain a deeper understanding of how helical cell morphology promotes host colonization by H. pylori, we used three-dimensional confocal microscopy to visualize the clinical isolate PMSS1 and an isogenic straight-rod mutant (Δcsd6) within thick longitudinal mouse stomach sections. We also performed volumetric image analysis to quantify the number of bacteria residing within corpus and antral glands in addition to measuring total CFU. We found that straight rods show attenuation during acute colonization of the stomach (1 day or 1 week postinfection) as measured by total CFU. Our quantitative imaging revealed that wild-type bacteria extensively colonized antral glands at 1 week postinfection, while csd6 mutants showed variable colonization of the antrum at this time point. During chronic infection (1 or 3 months postinfection), total CFU were highly variable but similar for wild-type and straight rods. Both wild-type and straight rods persisted and expanded in corpus glands during chronic infection. However, the straight rods showed reduced inflammation and disease progression. Thus, helical cell shape contributes to tissue interactions that promote inflammation during chronic infection, in addition to facilitating niche acquisition during acute infection.

The Helicobacter pylori cell shape promoting protein Csd5 interacts with the cell wall, MurF, and the bacterial cytoskeleton.

Chronic infection with Helicobacter pylori can lead to the development of gastric ulcers and stomach cancers. The helical cell shape of H. pylori promotes stomach colonization. Screens for loss of helical shape have identified several periplasmic peptidoglycan (PG) hydrolases and non-enzymatic putative scaffolding proteins, including Csd5. Both over and under expression of the PG hydrolases perturb helical shape, but the mechanism used to coordinate and localize their enzymatic activities is not known. Using immunoprecipitation and mass spectrometry we identified Csd5 interactions with cytosolic proteins CcmA, a bactofilin required for helical shape, and MurF, a PG precursor synthase, as well as the inner membrane spanning ATP synthase. A combination of Csd5 domain deletions, point mutations, and transmembrane domain chimeras revealed that the N-terminal transmembrane domain promotes MurF, CcmA, and ATP synthase interactions, while the C-terminal SH3 domain mediates PG binding. We conclude that Csd5 promotes helical shape as part of a membrane associated, multi-protein shape complex that includes interactions with the periplasmic cell wall, a PG precursor synthesis enzyme, the bacterial cytoskeleton, and ATP synthase.

Characterization of morphological conversion of Helicobacter pylori under anaerobic conditions.

Helicobacter pylori (H. pylori), a gram-negative microaerophilic bacterial pathogen that colonizes the stomachs of more than half of all humans, is linked to chronic gastritis, peptic ulcers and gastric cancer. Spiral-shaped H. pylori undergo morphologic conversion to a viable but not culturable coccoid form when they transit from the microaerobic stomach into the anaerobic intestinal tract. However, little is known about the morphological and pathogenic characteristics of H. pylori under prolonged anaerobic conditions. In this study, scanning electron microscopy was used to document anaerobiosis-induced morphological changes of H. pylori, from helical to coccoid to a newly defined fragmented form. Western blot analysis indicated that all three forms express certain pathogenic proteins, including the bacterial cytotoxin-associated gene A (CagA), components of the cag-Type IV secretion system (TFSS), the blood group antigen-binding adhesin BabA, and UreA (an apoenzyme of urease), almost equally. Similar urease activities were also detected in all three forms of H. pylori. However, in contrast to the helical form, bacterial motility and TFSS activity were found to have been abrogated in the anaerobiosis-induced coccoid and fragmented forms of H. pylori. Notably, it was demonstrated that some of the anaerobiosis-induced fragmented state cells could be converted to proliferation-competent helical bacteria in vitro. These results indicate that prolonged exposure to the anaerobic intestine may not eliminate the potential for H. pylori to revert to the helical pathogenic state.

The synthesis and evaluation of phenoxyacylhydroxamic acids as potential agents for Helicobacter pylori infections.

Two series of ω-phenoxy contained acylhydroxamic acids as novel urease inhibitors were designed and synthesized. Biological activity evaluations revealed that ω-phenoxypropinoylhydroxamic acids were more active than phenoxyacetohydroxamic acids. Out of these compounds, 3-(3,4-dichlorophenoxy)propionylhydroxamic acid c24 showed significant potency against urease in both cell free extract (IC50 = 0.061 ±â€¯0.003 µM) and intact cell (IC50 = 0.89 ±â€¯0.05 µM), being over 450- and 120-fold more potent than the clinically prescribed urease inhibitor AHA, repectively. Non-linear fitting of experimental data (V-[S]) suggested a mixed-type inhibition mechanism and a dual site binding mode of these compounds.

Erythromycin encapsulation in nanoemulsion-based delivery systems for treatment of Helicobacter pylori infection: Protection and synergy.

Poorly water-soluble and unstable compounds are difficult to develop as drug products using conventional formulation techniques. The aim of the present study was to develop and evaluate a nanoformulation prepared by a hot high-pressure homogenization method, which was a scalable and solvent-free process. We successfully prepared stable nanodispersions to protect a labile antibiotic, erythromycin. The mean diameter of the dispersed droplets was approximately 150 nm, and size distribution was unimodal. Dispersion was physically stable at room temperature for over six months. Using erythromycin as a model compound, we studied its antimicrobial activity in vitro on Helicobacter pylori. Results showed that drug encapsulation improves API stability in an acidic environment and is conducive to a synergistic effect between the drug and the formulation.

The Cell Shape-determining Csd6 Protein from Helicobacter pylori Constitutes a New Family of L,D-Carboxypeptidase.

Helicobacter pylori causes gastrointestinal diseases, including gastric cancer. Its high motility in the viscous gastric mucosa facilitates colonization of the human stomach and depends on the helical cell shape and the flagella. In H. pylori, Csd6 is one of the cell shape-determining proteins that play key roles in alteration of cross-linking or by trimming of peptidoglycan muropeptides. Csd6 is also involved in deglycosylation of the flagellar protein FlaA. To better understand its function, biochemical, biophysical, and structural characterizations were carried out. We show that Csd6 has a three-domain architecture and exists as a dimer in solution. The N-terminal domain plays a key role in dimerization. The middle catalytic domain resembles those of l,d-transpeptidases, but its pocket-shaped active site is uniquely defined by the four loops I to IV, among which loops I and III show the most distinct variations from the known l,d-transpeptidases. Mass analyses confirm that Csd6 functions only as an l,d-carboxypeptidase and not as an l,d-transpeptidase. The d-Ala-complexed structure suggests possible binding modes of both the substrate and product to the catalytic domain. The C-terminal nuclear transport factor 2-like domain possesses a deep pocket for possible binding of pseudaminic acid, and in silico docking supports its role in deglycosylation of flagellin. On the basis of these findings, it is proposed that H. pylori Csd6 and its homologs constitute a new family of l,d-carboxypeptidase. This work provides insights into the function of Csd6 in regulating the helical cell shape and motility of H. pylori.

Helical shape of Helicobacter pylori requires an atypical glutamine as a zinc ligand in the carboxypeptidase Csd4.

Peptidoglycan modifying carboxypeptidases (CPs) are important determinants of bacterial cell shape. Here, we report crystal structures of Csd4, a three-domain protein from the human gastric pathogen Helicobacter pylori. The catalytic zinc in Csd4 is coordinated by a rare His-Glu-Gln configuration that is conserved among most Csd4 homologs, which form a distinct subfamily of CPs. Substitution of the glutamine to histidine, the residue found in prototypical zinc carboxypeptidases, resulted in decreased enzyme activity and inhibition by phosphate. Expression of the histidine variant at the native locus in a H. pylori csd4 deletion strain did not restore the wild-type helical morphology. Biochemical assays show that Csd4 can cleave a tripeptide peptidoglycan substrate analog to release m-DAP. Structures of Csd4 with this substrate analog or product bound at the active site reveal determinants of peptidoglycan specificity and the mechanism to cleave an isopeptide bond to release m-DAP. Our data suggest that Csd4 is the archetype of a new CP subfamily with a domain scheme that differs from this large family of peptide-cleaving enzymes.

Helicobacter pylori CheZ(HP) and ChePep form a novel chemotaxis-regulatory complex distinct from the core chemotaxis signaling proteins and the flagellar motor.

Chemotaxis is important for Helicobacter pylori to colonize the stomach. Like other bacteria, H. pylori uses chemoreceptors and conserved chemotaxis proteins to phosphorylate the flagellar rotational response regulator, CheY, and modulate the flagellar rotational direction. Phosphorylated CheY is returned to its non-phosphorylated state by phosphatases such as CheZ. In previously studied cases, chemotaxis phosphatases localize to the cellular poles by interactions with either the CheA chemotaxis kinase or flagellar motor proteins. We report here that the H. pylori CheZ, CheZ(HP), localizes to the poles independently of the flagellar motor, CheA, and all typical chemotaxis proteins. Instead, CheZ(HP) localization depends on the chemotaxis regulatory protein ChePep, and reciprocally, ChePep requires CheZ(HP) for its polar localization. We furthermore show that these proteins interact directly. Functional domain mapping of CheZ(HP) determined the polar localization motif lies within the central domain of the protein and that the protein has regions outside of the active site that participate in chemotaxis. Our results suggest that CheZ(HP) and ChePep form a distinct complex. These results therefore suggest the intriguing idea that some phosphatases localize independently of the other chemotaxis and motility proteins, possibly to confer unique regulation on these proteins' activities.

Negative Effect of Proton-pump Inhibitors (PPIs) on Helicobacter pylori Growth, Morphology, and Urease Test and Recovery after PPI Removal--An In vitro Study.

BACKGROUND: Proton-pump inhibitor (PPI) consumption does lead to false-negative results of Helicobacter pylori diagnostic tests such as biopsy culture and rapid urease test (RUT). MATERIALS AND METHODS: Helicobacter pylori isolates from 112 dyspeptic patients with (56.5%) or without (43.5%) PPI consumption were recruited for examining the negative effects of omeprazole (OMP), lansoprazole (LPZ), and pantoprazole (PAN) on H. pylori viability, morphology, and urease, in vitro. The effect of a sublethal concentration of OMP on bacterial features and their recovery after removal of OMP was also assessed. RESULTS: Of 112 culture-positive gastric biopsies, 87.5% were RUT positive and 12.5% RUT negative. There was a significant correlation between negative RUT results and PPI consumption (p < .05). OMP (minimum inhibitory concentration, MIC 32 µg/mL) and LPZ (MIC 8 µg/mL) inhibited the growth of 78.6% of H. pylori isolates. OMP and LPZ inhibited urease of 90.3% of isolates between 0 and 40 minutes and 54.4% between 20 and 40 minutes, respectively. PAN did not inhibit H. pylori growth and urease. Three 3-day (9 days) consecutive subcultures of H. pylori on brucella blood agar (BBA) supplemented with OMP resulted in reduced bacterial viability (1+), compared with control (4+), change of spiral morphology to coccoid, and reduction in pink color intensity in urea agar. Bacterial growth (1+), morphology, and urease test did not improve after the first 3-day and second 3-day (6 days) subcultures on BBA. However, relative recovery occurred after the third 3-day (9 days) subculture and complete recovery was observed after the fourth 3-day (12 days) subculture, as confluent growth (4+), 100% spiral cells, and strong urease test. CONCLUSION: Proton-pump Inhibitors exert transient negative effects on H. pylori viability, morphology, and urease test. Accordingly, cessation of PPI consumption at least 12 days before endoscopy could help avoiding false-negative results of H. pylori diagnostic tests.

Regulation of Helicobacter pylori Virulence Within the Context of Iron Deficiency.

Helicobacter pylori strains that harbor the oncoprotein CagA increase gastric cancer risk, and this risk is augmented under iron-deficient conditions. We demonstrate here that iron depletion induces coccoid morphology in strains lacking cagA. To evaluate the stability of augmented H. pylori virulence phenotypes stimulated by low-iron conditions, H. pylori isolated from iron-depleted conditions in vivo were serially passaged in vitro. Long-term passage decreased the ability of hypervirulent strains to translocate CagA or induce interleukin 8, indicating that hypervirulent phenotypes stimulated by low-level iron conditions are reversible. Therefore, rectifying iron deficiency may attenuate disease among H. pylori-infected persons with no response to antibiotics.

Probing cell-surface carbohydrate binding proteins with dual-modal glycan-conjugated nanoparticles.

Dual-modal fluorescent magnetic glyconanoparticles have been prepared and shown to be powerful in probing lectins displayed on pathogenic and mammalian cell surfaces. Blood group H1- and Le(b)-conjugated nanoparticles were found to bind to BabA displaying Helicobacter pylori, and Le(a)- and Le(b)-modified nanoparticles are both recognized by and internalized into DC-SIGN and SIGN-R1 expressing mammalian cells via lectin-mediated endocytosis. In addition, glyconanoparticles block adhesion of H. pylori to mammalian cells, suggesting that they can serve as inhibitors of infection of host cells by this pathogen. It has been also shown that owing to their magnetic properties, glyconanoparticles are useful tools to enrich lectin expressing cells. The combined results indicate that dual-modal glyconanoparticles are biocompatible and that they can be employed in lectin-associated biological studies and biomedical applications.

Animal Model Reveals Potential Waterborne Transmission of Helicobacter pylori Infection.

BACKGROUND: Helicobacter pylori infection has been consistently associated with lack of access to clean water and proper sanitation, but no studies have demonstrated that the transmission of H. pylori can occur from drinking contaminated water. In this study, we used a laboratory mouse model to test whether waterborne H. pylori could cause gastric infection. MATERIALS AND METHODS: Groups of immunocompetent C57/BL6 Helicobacter-free mice were exposed to static concentrations (1.29 × 10(5), 10(6), 10(7), 10(8), and 10(9) CFU/L) of H. pylori in their drinking water for 4 weeks. One group of Helicobacter-free mice was exposed to uncontaminated water as a negative control. H. pylori morphology changes in water were examined using microscopy Live/Dead staining. Following exposure, H. pylori infection and inflammation status in the stomach were evaluated using quantitative culture, PCR, the rapid urease test, and histology. RESULTS: None of the mice in the negative control or 10(5) groups were infected. One of 20 cages (one of 40 mice) of the 10(6) group, three of 19 cages (four of 38 mice) of the 10(7) CFU/L group, 19 of 20 cages (33 of 40 mice) of the 10(8) group, and 20 of 20 cages (39 of 40 mice) of the 10(9) CFU/L group were infected. Infected mice had significantly higher gastric inflammation than uninfected mice (27.86% higher inflammation, p < .0001). CONCLUSIONS: We offer proof that H. pylori in water is infectious in mice, suggesting that humans drinking contaminated water may be at risk of contracting H. pylori infection. Much work needs to be performed to better understand the risk of infection from drinking H. pylori-contaminated water.

Intracellular locations of replication proteins and the origin of replication during chromosome duplication in the slowly growing human pathogen Helicobacter pylori.

We followed the position of the replication complex in the pathogenic bacterium Helicobacter pylori using antibodies raised against the single-stranded DNA binding protein (HpSSB) and the replicative helicase (HpDnaB). The position of the replication origin, oriC, was also localized in growing cells by fluorescence in situ hybridization (FISH) with fluorescence-labeled DNA sequences adjacent to the origin. The replisome assembled at oriC near one of the cell poles, and the two forks moved together toward the cell center as replication progressed in the growing cell. Termination and resolution of the forks occurred near midcell, on one side of the septal membrane. The duplicated copies of oriC did not separate until late in elongation, when the daughter chromosomes segregated into bilobed nucleoids, suggesting sister chromatid cohesion at or near the oriC region. Components of the replication machinery, viz., HpDnaB and HpDnaG (DNA primase), were found associated with the cell membrane. A model for the assembly and location of the H. pylori replication machinery during chromosomal duplication is presented.

Flow cytometry-based enrichment for cell shape mutants identifies multiple genes that influence Helicobacter pylori morphology.

The helical cell shape of Helicobacter pylori is highly conserved and contributes to its ability to swim through and colonize the viscous gastric mucus layer. A multi-faceted peptidoglycan (PG) modification programme involving four recently characterized peptidases and two accessory proteins is essential for maintaining H. pylori's helicity. To expedite identification of additional shape-determining genes, we employed flow cytometry with fluorescence-activated cell sorting (FACS) to enrich a transposon library for bacterial cells with altered light scattering profiles that correlate with perturbed cell morphology. After a single round of sorting, 15% of our clones exhibited a stable cell shape defect, reflecting 37-fold enrichment. Sorted clones with straight rod morphology contained insertions in known PG peptidases, as well as an insertion in csd6, which we demonstrated has ld-carboxypeptidase activity and cleaves monomeric tetrapeptides in the PG sacculus, yielding tripeptides. Other mutants had only slight changes in helicity due to insertions in genes encoding MviN/MurJ, a protein possibly involved in initiating PG synthesis, and the hypothetical protein HPG27_782. Our findings demonstrate FACS robustly detects perturbations of bacterial cell shape and identify additional PG peptide modifications associated with helical cell shape in H. pylori.