Counterclockwise rotation of the flagellum promotes biofilm initiation in Helicobacter pylori.

Motility promotes biofilm initiation during the early steps of this process: microbial surface association and attachment. Motility is controlled in part by chemotaxis signaling, so it seems reasonable that chemotaxis may also affect biofilm formation. There is a gap, however, in our understanding of the interactions between chemotaxis and biofilm formation, partly because most studies analyzed the phenotype of only a single chemotaxis signaling mutant, e.g., cheA. Here, we addressed the role of chemotaxis in biofilm formation using a full set of chemotaxis signaling mutants in Helicobacter pylori, a class I carcinogen that infects more than half the world's population and forms biofilms. Using mutants that lack each chemotaxis signaling protein, we found that chemotaxis signaling affected the biofilm initiation stage, but not mature biofilm formation. Surprisingly, some chemotaxis mutants elevated biofilm initiation, while others inhibited it in a manner that was not tied to chemotaxis ability or ligand input. Instead, the biofilm phenotype correlated with flagellar rotational bias. Specifically, mutants with a counterclockwise bias promoted biofilm initiation, e.g., ∆cheA, ∆cheW, or ∆cheV1; in contrast, those with a clockwise bias inhibited it, e.g., ∆cheZ, ∆chePep, or ∆cheV3. We tested this correlation using a counterclockwise bias-locked flagellum, which induced biofilm formation independent of the chemotaxis system. These CCW flagella, however, were not sufficient to induce biofilm formation, suggesting there are downstream players. Overall, our work highlights the new finding that flagellar rotational direction promotes biofilm initiation, with the chemotaxis signaling system operating as one mechanism to control flagellar rotation. IMPORTANCE: Chemotaxis signaling systems have been reported to contribute to biofilm formation in many bacteria; however, how they regulate biofilm formation remains largely unknown. Chemotaxis systems are composed of many distinct kinds of proteins, but most previous work analyzed the biofilm effect of loss of only a few. Here, we explored chemotaxis' role during biofilm formation in the human-associated pathogenic bacterium Helicobacter pylori. We found that chemotaxis proteins are involved in biofilm initiation in a manner that correlated with how they affected flagellar rotation. Biofilm initiation was high in mutants with counterclockwise (CCW) flagellar bias and low in those with clockwise bias. We supported the idea that a major driver of biofilm formation is flagellar rotational direction using a CCW-locked flagellar mutant, which stays CCW independent of chemotaxis input and showed elevated biofilm initiation. Our data suggest that CCW-rotating flagella, independent of chemotaxis inputs, are a biofilm-promoting signal.

Helicobacter pylori cheV1 mutants recover semisolid agar migration due to loss of a previously uncharacterized Type IV filament membrane alignment complex homolog.

The bacterial chemotaxis system is a well-understood signaling pathway that promotes bacterial success. Chemotaxis systems comprise chemoreceptors and the CheA kinase, linked by CheW or CheV scaffold proteins. Scaffold proteins provide connections between chemoreceptors and CheA and also between chemoreceptors to create macromolecular arrays. Chemotaxis is required for host colonization by many microbes, including the stomach pathogen Helicobacter pylori. This bacterium builds chemoreceptor-CheA contacts with two distinct scaffold proteins, CheW and CheV1. H. pylori cheW or cheV1 deletion mutants both lose chemoreceptor array formation, but show differing semisolid agar chemotaxis assay behaviors: ∆cheW mutants exhibit total migration failure, whereas ∆cheV1::cat mutants display a 50% reduction. On investigating these varied responses, we found that both mutants initially struggle with migration. However, over time, ∆cheV1::cat mutants develop a stable, enhanced migration capability, termed "migration-able" (Mig+). Whole-genome sequencing analysis of four distinct ∆cheV1::cat Mig+ strains identified single-nucleotide polymorphisms (SNPs) in hpg27_252 (hp0273) that were predicted to truncate the encoded protein. Computational analysis of the hpg27_252-encoded protein revealed it encoded a hypothetical protein that was a remote homolog of the PilO Type IV filament membrane alignment complex protein. Although H. pylori lacks Type IV filaments, our analysis showed it retains an operon of genes for homologs of PilO, PilN, and PilM. Deleting hpg27_252 in the ∆cheV1::cat or wild type strain resulted in enhanced migration in semisolid agar. Our study thus reveals that while cheV1 mutants initially have significant migration defects, they can recover the migration ability through genetic suppressors, highlighting a complex regulatory mechanism in bacterial migration. IMPORTANCE: Chemotactic motility, present in over half of bacteria, depends on chemotaxis signaling systems comprising receptors, kinases, and scaffold proteins. In Helicobacter pylori, a stomach pathogen, chemotaxis is crucial for colonization, with CheV1 and CheW as key scaffold proteins. While both scaffolds are essential for building chemoreceptor complexes, their roles vary in other assays. Our research reexamines cheV1 mutants' behavior in semisolid agar, a standard chemotaxis test. Initially, cheV1 mutants exhibited defects similar to those of cheW mutants, but they evolved genetic suppressors that enhanced migration. These suppressors involve mutations in a previously uncharacterized gene, unknown in motility behavior. Our findings highlight the significant chemotaxis defects in cheV1 mutants and identify new elements influencing bacterial motility.

Bacterial flagella hijack type IV pili proteins to control motility.

Bacterial flagella and type IV pili (TFP) are surface appendages that enable motility and mechanosensing through distinct mechanisms. These structures were previously thought to have no components in common. Here, we report that TFP and some flagella share proteins PilO, PilN, and PilM, which we identified as part of the Helicobacter pylori flagellar motor. H. pylori mutants lacking PilO or PilN migrated better than wild type in semisolid agar because they continued swimming rather than aggregated into microcolonies, mimicking the TFP-regulated surface response. Like their TFP homologs, flagellar PilO/PilN heterodimers formed a peripheral cage that encircled the flagellar motor. These results indicate that PilO and PilN act similarly in flagella and TFP by differentially regulating motility and microcolony formation when bacteria encounter surfaces.

Altering under-represented DNA sequences elevates bacterial transformation efficiency.

A cornerstone of bacterial molecular biology is the ability to genetically manipulate the microbe under study. Many bacteria are difficult to manipulate genetically, a phenotype due in part to robust removal of newly acquired DNA, for example, by restriction-modification (R-M) systems. Here, we report approaches that dramatically improve bacterial transformation efficiency, piloted using a microbe that is challenging to transform due to expression of many R-M systems, Helicobacter pylori. Initially, we identified conditions that dampened expression of several R-M systems and concomitantly enhanced transformation efficiency. We then identified an approach that would broadly protect newly acquired DNA. We computationally predicted under-represented short DNA sequences in the H. pylori genome, with the idea that these sequences reflect targets of sequence-based surveillance such as R-M systems. We then used this information to modify and eliminate such sites in antibiotic resistance cassettes, creating a "stealth" version. Modifying antibiotic resistance cassettes in this way resulted in significantly higher transformation efficiency compared to non-modified cassettes, a response that was genomic loci independent. Our results suggest that avoiding R-M systems, via modification of under-represented DNA sequences or transformation conditions, is a powerful method to enhance DNA transformation. Our approach to identify under-represented sequences is applicable to any microbe with a sequenced genome.IMPORTANCEManipulating the genomes of bacteria is critical to many fields. Such manipulations are made by genetic engineering, which often requires new pieces of DNA to be added to the genome. Bacteria have robust systems for identifying and degrading new DNA, some of which rely on restriction enzymes. These enzymes cut DNA at specific sequences. We identified a set of DNA sequences that are missing normally from a bacterium's genome, more than would be expected by chance. Eliminating these sequences from a new piece of DNA allowed it to be incorporated into the bacterial genome at a higher frequency than new DNA containing the sequences. Removing such sequences appears to allow the new DNA to fly under the bacterial radar in "stealth" mode. This transformation improvement approach is straightforward to apply and likely broadly applicable.

Discovery of Type IV filament membrane alignment complex homologs in H. pylori that promote soft-agar migration.

The stomach pathogen Helicobacter pylori utilizes two scaffold proteins, CheW and CheV1, to build critical chemotaxis arrays. Chemotaxis helps bacteria establish and maintain infection. Mutants lacking either of these chemotaxis proteins have different soft agar phenotypes: deletion of cheW creates non-chemotactic strains, while deletion of cheV1 results in 50% loss of chemotaxis. In this work, we characterized the cheV1 deletion mutant phenotype in detail. cheV1 deletion mutants had poor soft-agar migration initially, but regained migration ability over time. This improved bacterial migration was stable, suggesting a genetic suppressor phenotype, termed Che+. Whole-genome sequencing analysis of four distinct cheV1 Che+ strains revealed single nucleotide polymorphisms (SNPs) in a common gene, HPG27_252 (HP0273). These SNPs were predicted to truncate the encoded protein. To confirm the role of HPG27_252 in the cheV1 phenotype, we created a targeted deletion of HPG27_252 and found that loss of HPG27_252 enhanced soft-agar migration. HPG27_252 and CheV1 appear to interact directly, based on bacterial two-hybrid analysis. HPG27_252 is predicted to encode a 179 amino acid, 21 kDa protein annotated as a hypothetical protein. Computational analysis revealed this protein to be a remote homolog of the PilO Type IV filament membrane alignment complex protein. Although H. pylori is not known to possess Type IV filaments, our analysis showed it retains an operon of genes for homologs of PilO, PilN, and PilM, but does not possess other Type IV pili genes. Our data suggest the PilO homolog plays a role in regulating H. pylori chemotaxis and motility, suggesting new ideas about evolutionary steps for controlling migration through semi-solid media.

Helicobacter pylori initiates successful gastric colonization by utilizing L-lactate to promote complement resistance.

The complement system has long been appreciated for its role in bloodborne infections, but its activities in other places, including the gastrointestinal tract, remain elusive. Here, we report that complement restricts gastric infection by the pathogen Helicobacter pylori. This bacterium colonized complement-deficient mice to higher levels than wild-type counterparts, particularly in the gastric corpus region. H. pylori uses uptake of the host molecule L-lactate to create a complement-resistant state that relies on blocking the deposition of the active complement C4b component on H. pylori's surface. H. pylori mutants unable to achieve this complement-resistant state have a significant mouse colonization defect that is largely corrected by mutational removal of complement. This work highlights a previously unknown role for complement in the stomach, and has revealed an unrecognized mechanism for microbial-derived complement resistance.

FliL Functions in Diverse Microbes to Negatively Modulate Motor Output via Its N-Terminal Region.

The flagellar motor protein FliL is conserved across many microbes, but its exact role has been obscured by varying fliL mutant phenotypes. We reanalyzed results from fliL studies and found they utilized alleles that differed in the amount of N- and C-terminal regions that were retained. Alleles that retain the N-terminal cytoplasmic and transmembrane helix (TM) regions in the absence of the C-terminal periplasmic domain result in loss of motility, while alleles that completely lack the N-terminal region, independent of the periplasmic domain, retain motility. We then tested this prediction in Helicobacter pylori fliL and found support for the idea. This analysis suggests that FliL function may be more conserved across bacteria than previously thought, that it is not essential for motility, and that the N-terminal region has the negative ability to regulate motor function. IMPORTANCE FliL is a protein found in the flagellar motor of bacteria, but what it does was not clear. To study FliL function, scientists often remove it and see what happens. Loss of FliL was thought to have different effects depending on the microbe. We uncovered, however, that part of the confusion arose because scientists inadvertently removed different parts of the protein. Our analysis and data suggest that leaving the N-terminal regions blocks motility, while fully removing FliL allows normal motility. This finding will help scientists understand FliL because it clarifies what needs to be removed to fully eliminate the protein, and also that the N-terminal region can block motility.

Helicobacter pylori Chronic-Stage Inflammation Undergoes Fluctuations That Are Altered in tlpA Mutants.

Helicobacter pylori colonizes half of the world's population and is responsible for a significant disease burden by causing gastritis, peptic ulcers, and gastric cancer. The development of host inflammation drives these diseases, but there are still open questions in the field about how H. pylori controls this process. We characterized H. pylori inflammation using an 8-month mouse infection time course and comparison of the wild type (WT) and a previously identified mutant lacking the TlpA chemoreceptor that causes elevated inflammation. Our work shows that H. pylori chronic-stage corpus inflammation undergoes surprising fluctuations, with changes in Th17 and eosinophil numbers. The H. pylori tlpA mutant changed the inflammation temporal characteristics, resulting in different inflammation from the wild type at some time points. tlpA mutants have equivalent total and gland colonization in late-stage infections. During early infection, in contrast, they show elevated gland and total colonization compared to those by WT. Our results suggest the chronic inflammation setting is dynamic and may be influenced by colonization properties of early infection.

Methylation-Independent Chemotaxis Systems Are the Norm for Gastric-Colonizing Helicobacter Species.

Many bacteria and archaea rely on chemotaxis signal transduction systems for optimal fitness. These complex, multiprotein signaling systems have core components found in all chemotactic microbes, as well as variable proteins found in only some species. We do not yet understand why these variations exist or whether there are specific niches that favor particular chemotaxis signaling organization. One variation is in the presence/absence of the chemotaxis methylation adaptation enzymes CheB and CheR. Genes for CheB and CheR are missing in the gastric pathogen Helicobacter pylori but present in related Helicobacter that colonize the liver or intestine. In this work, we asked whether there was a general pattern of CheB/CheR across multiple Helicobacter species. Helicobacter spp. all possess chemotactic behavior, based on the presence of genes for core signaling proteins CheA, CheW, and chemoreceptors. Genes for the CheB and CheR proteins, in contrast, were variably present. Niche mapping supported the idea that these genes were present in enterohepatic Helicobacter species and absent in gastric ones. We then analyzed whether there were differences between gastric and enterohepatic species in the CheB/CheR chemoreceptor target methylation sites. Indeed, these sites were less conserved in gastric species that lack CheB/CheR. Lastly, we determined that cheB and cheR could serve as markers to indicate whether an unknown Helicobacter species was of enterohepatic or gastric origin. Overall, these findings suggest the interesting idea that methylation-based adaptation is not required in specific environments, particularly the stomach. IMPORTANCE Chemotaxis signal transduction systems are common in the archaeal and bacterial world, but not all systems contain the same components. The rationale for this system variation remains unknown. In this report, comparative genomics analysis showed that the presence/absence of CheR and CheB is one main variation within the Helicobacter genus, and it is strongly associated with the niche of Helicobacter species: gastric Helicobacter species, which infect animal stomachs, have lost their CheB and CheR, while enterohepatic Helicobacter species, which infect the liver and intestine, retain them. This study not only provides an example that a chemotaxis system variant is associated with particular niches but also proposes that CheB and CheR are new markers distinguishing gastric from enterohepatic Helicobacter species.

The flagellar motor protein FliL forms a scaffold of circumferentially positioned rings required for stator activation.

The flagellar motor stator is an ion channel nanomachine that assembles as a ring of the MotA5MotB2 units at the flagellar base. The role of accessory proteins required for stator assembly and activation remains largely enigmatic. Here, we show that one such assembly factor, the conserved protein FliL, forms an integral part of the Helicobacter pylori flagellar motor in a position that colocalizes with the stator. Cryogenic electron tomography reconstructions of the intact motor in whole wild-type cells and cells lacking FliL revealed that the periplasmic domain of FliL (FliL-C) forms 18 circumferentially positioned rings integrated with the 18 MotAB units. FliL-C formed partial rings in the crystal, and the crystal structure-based full ring model was consistent with the shape of the rings observed in situ. Our data suggest that each FliL ring is coaxially sandwiched between the MotA ring and the dimeric periplasmic MotB moiety of the stator unit and that the central hole of the FliL ring has density that is consistent with the plug/linker region of MotB in its extended, active conformation. Significant structural similarities were found between FliL-C and stomatin/prohibitin/flotillin/HflK/C domains of scaffolding proteins, suggesting that FliL acts as a scaffold. The binding energy released upon association of FliL with the stator units could be used to power the release of the plug helices. The finding that isolated FliL-C forms stable partial rings provides an insight into the putative mechanism by which the FliL rings assemble around the stator units.

The dCache Chemoreceptor TlpA of Helicobacter pylori Binds Multiple Attractant and Antagonistic Ligands via Distinct Sites.

The Helicobacter pylori chemoreceptor TlpA plays a role in dampening host inflammation during chronic stomach colonization. TlpA has a periplasmic dCache_1 domain, a structure that is capable of sensing many ligands; however, the only characterized TlpA signals are arginine, bicarbonate, and acid. To increase our understanding of TlpA's sensing profile, we screened for diverse TlpA ligands using ligand binding arrays. TlpA bound seven ligands with affinities in the low- to middle-micromolar ranges. Three of these ligands, arginine, fumarate, and cysteine, were TlpA-dependent chemoattractants, while the others elicited no response. Molecular docking experiments, site-directed point mutants, and competition surface plasmon resonance binding assays suggested that TlpA binds ligands via both the membrane-distal and -proximal dCache_1 binding pockets. Surprisingly, one of the nonactive ligands, glucosamine, acted as a chemotaxis antagonist, preventing the chemotaxis response to chemoattractant ligands, and acted to block the binding of ligands irrespective of whether they bound the membrane-distal or -proximal dCache_1 subdomains. In total, these results suggest that TlpA senses multiple attractant ligands as well as antagonist ones, an emerging theme in chemotaxis systems. IMPORTANCE Numerous chemotactic bacterial pathogens depend on the ability to sense a diverse array of signals through chemoreceptors to achieve successful colonization and virulence within their host. The signals sensed by chemoreceptors, however, are not always fully understood. This is the case for TlpA, a dCache_1 chemoreceptor of H. pylori that enables the bacterium to induce less inflammation during chronic infections. H. pylori causes a significant global disease burden, which is driven by the development of gastric inflammation. Accordingly, it is essential to understand the processes by which H. pylori modulates host inflammation. This work uncovers the signals that TlpA can sense and highlights the underappreciated ability to regulate chemotactic responses by antagonistic chemoreceptor ligands, which is an emerging theme among other chemotactic systems.

Gastric Metabolomics Detects Helicobacter pylori Correlated Loss of Numerous Metabolites in Both the Corpus and Antrum.

Helicobacter pylori is a chronic bacterial pathogen that thrives in several regions of the stomach, causing inflammation that can vary by site and result in distinct disease outcomes. Whether the regions differ in terms of host-derived metabolites is not known. We thus characterized the regional variation of the metabolomes of mouse gastric corpus and antrum organoids and tissue. The uninfected secreted organoid metabolites differed between the corpus and antrum in only seven metabolites as follows: lactic acid, malic acid, phosphoethanolamine, alanine, uridine, glycerol, and isoleucine. Several of the secreted chemicals were depleted upon H. pylori infection in both regions, including urea, cholesterol, glutamine, fumaric acid, lactic acid, citric acid, malic acid, and multiple nonessential amino acids. These results suggest a model in which H. pylori preferentially uses carboxylic acids and amino acids in complex environments, and these are found in both the corpus and antrum. When organoid metabolites were compared to mouse tissue, there was little overlap. The tissue corpus and antrum metabolomes were distinct, including antrum-elevated 5-methoxytryptamine, lactic acid, and caprylic acid, and corpus-elevated phospholipid products. The corpus and antrum remained distinct over an 8-month infection time course. The antrum displayed no significant changes between the time points in contrast to the corpus, which exhibited metabolite changes that were consistent with stress, tissue damage, and depletion of key nutrients, such as glutamine and fructose-6-phosphate. Overall, our results suggest that the corpus and antrum have largely but not completely overlapping metabolomes that change moderately upon H. pylori infection.

Genetic requirements and transcriptomics of Helicobacter pylori biofilm formation on abiotic and biotic surfaces.

Biofilm growth is a widespread mechanism that protects bacteria against harsh environments, antimicrobials, and immune responses. These types of conditions challenge chronic colonizers such as Helicobacter pylori but it is not fully understood how H. pylori biofilm growth is defined and its impact on H. pylori survival. To provide insights into H. pylori biofilm growth properties, we characterized biofilm formation on abiotic and biotic surfaces, identified genes required for biofilm formation, and defined the biofilm-associated gene expression of the laboratory model H. pylori strain G27. We report that H. pylori G27 forms biofilms with a high biomass and complex flagella-filled 3D structures on both plastic and gastric epithelial cells. Using a screen for biofilm-defective mutants and transcriptomics, we discovered that biofilm cells demonstrated lower transcripts for TCA cycle enzymes but higher ones for flagellar formation, two type four secretion systems, hydrogenase, and acetone metabolism. We confirmed that biofilm formation requires flagella, hydrogenase, and acetone metabolism on both abiotic and biotic surfaces. Altogether, these data suggest that H. pylori is capable of adjusting its phenotype when grown as biofilm, changing its metabolism, and re-shaping flagella, typically locomotion organelles, into adhesive structures.

Helicobacter pylori Biofilm Confers Antibiotic Tolerance in Part via A Protein-Dependent Mechanism.

Helicobacter pylori, a WHO class I carcinogen, is one of the most successful human pathogens colonizing the stomach of over 4.4 billion of the world's population. Antibiotic therapy represents the best solution but poor response rates have hampered the elimination of H. pylori. A growing body of evidence suggests that H. pylori forms biofilms, but the role of this growth mode in infection remains elusive. Here, we demonstrate that H. pylori cells within a biofilm are tolerant to multiple antibiotics in a manner that depends partially on extracellular proteins. Biofilm-forming cells were tolerant to multiple antibiotics that target distinct pathways, including amoxicillin, clarithromycin, and tetracycline. Furthermore, this tolerance was significantly dampened following proteinase K treatment. These data suggest that H. pylori adapts its phenotype during biofilm growth resulting in decreased antibiotic susceptibility but this tolerance can be partially ameliorated by extracellular protease treatment.

Helicobacter pylori Uses the TlpB Receptor To Sense Sites of Gastric Injury.

Helicobacter pylori is a pathogen that chronically colonizes the stomachs of approximately half of the world's population and contributes to the development of gastric inflammation. We demonstrated previously in vivo that H. pylori uses motility to preferentially colonize injury sites in the mouse stomach. However, the chemoreceptor responsible for sensing gastric injury has not yet been identified. In this study, we utilized murine gastric organoids (gastroids) and mutant H. pylori strains to investigate the components necessary for H. pylori chemotaxis. High-intensity 730-nm light (two-photon photodamage) was used to cause single-cell damage in gastroids, and repair of the damage was monitored over time; complete repair occurred within ∼10 min in uninfected gastroids. Wild-type H. pylori accumulated at the damage site after gastric damage induction. In contrast, mutants lacking motility (ΔmotB) or chemotaxis (ΔcheY) did not accumulate at the injury site. Using mutants lacking individual chemoreceptors, we found that only TlpB was required for H. pylori accumulation, while TlpA, TlpC, and TlpD were dispensable. All strains that were able to accumulate at the damage site limited repair. When urea (an identified chemoattractant sensed by TlpB) was microinjected into the gastroid lumen, it prevented the accumulation of H. pylori at damage sites. Overall, our findings demonstrate that H. pylori colonizes and limits repair at damage sites via chemotactic motility that requires the TlpB chemoreceptor to sense signals generated by gastric epithelial cells.