Biology subjects

Ottemann, K. M.

Publications and source records attributed to Ottemann, K. M..

8 recordsLinked to original sources

bioRxiv2025

Distinct flagellins differentially fine tune biofilm initiation via flagellar stator-associated proteins

Helicobacter pylori relies on flagellar motility to colonize the gastric niche. While H. pylori flagella serve multiple functions, including swimming motility, it has recently been appreciated that flagellar basal body properties also contribute to biofilm initiation. The H. pylori flagellar filament contains two spatially segregated flagellins, FlaA and FlaB, but their roles in the biofilm initiation process remained undefined. Here, we confirmed that both FlaA and FlaB are required for optimal motility, but found that they exert opposite effects on biofilm initiation: cells without FlaA display low biofilm initiation, while cells without FlaB display elevated initiation. To understand the molecular basis for this divergence, cryo-electron microscopy (cryo-EM) analysis of native flagellar filaments revealed that FlaA and FlaB have distinct supercoiled waveforms. We confirmed these intrinsic waveform curvatures by analyzing filaments from {Delta}flaA and {Delta}flaB mutants, providing a physical basis for their functional specialization. Genetic epistasis experiments further demonstrated that the enhanced early biofilm formation of the {Delta}flaB mutant depends on PilO, a stator-associated component of the flagellar motor recently shown to drive a reciprocal biofilm and motility response. Our findings establish that H. pylori has developed functionally specialized flagellins that work with motor-dependent signaling to dynamically balance surface colonization and motility.

microbiology↗

bioRxiv2024

The C-terminus of the TlpD cytoplasmic chemoreceptor promotes polar localization and function

Bacteria localize proteins to distinct subcellular locations, including chemoreceptors, which frequently localize to the bacterial pole. Although some polarity-promoting mechanisms have been described, many chemoreceptors lack clear routes to becoming polar. TlpD of the bacterial pathogen Helicobacter pylori is one such protein. This cytoplasmic chemoreceptor localizes to the pole in a manner that is independent of the other chemoreceptors. In this work, we evaluated the role of TlpD domains in its function. Truncated proteins were created that lacked different amounts of the N- or C-termini and expressed in H. pylori in place of native tlpD or as the sole chemoreceptor. These TlpD variants were examined for expression, protein localization, association with chemotaxis signaling proteins, and effect on motility. TlpD that lacked any portion of the N-terminal 104 amino acids did not produce detectable protein. In contrast, TlpD retained expression with loss of the C-terminal 45 amino acids. TlpD lacking the last 45 amino acids (TlpD{Delta}C4) preserved the ability to interact with CheW and CheV proteins based on bacterial two-hybrid analysis but were unable to localize to the pole either on their own or in the presence of other chemoreceptors. TlpD{Delta}C4 was found diffuse in the cytoplasm, and interacted with CheV1, CheV2, and CheV3 at this location but not CheW. TlpD{Delta}C4 did not confer chemotactic abilities in soft agar chemotaxis assays. These findings suggest the C-terminal end of TlpD plays a previously unappreciated role in promoting TlpD polar localization and function. ImportanceBacteria place their proteins in specific locations that are required for the proteins to function including the bacterial pole. How the bacterial cell identifies which proteins go to the pole is not fully understood. In this work, we dissect parts of a protein called TlpD that naturally goes to the pole. We find that mutants lacking one end of TlpD lose their polar placement, but retain other abilities. TlpD allows directed motility known as chemotaxis. This ability is critical for infection in H. pylori and numerous other pathogens. When TlpD loses its polar placement, the protein no longer functions for chemotaxis, laying the foundation for future studies that can dissect how this segment promotes function and eventually translate into therapies for H. pylori infection.

microbiology↗

bioRxiv2022

Helicobacter chronic stage inflammation undergoes fluctuations that are altered in tlpA mutants

Helicobacter pylori colonizes half of the worlds 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 eight month mouse infection time course and comparison of wild type 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 wild type at some time points. tlpA mutants have equivalent total and gland colonization at late stage infections. During early infection, in contrast, they show elevated gland and total colonization compared to WT. Our results suggest the chronic inflammation setting is dynamic, and may be influenced by colonization properties of early infection. ImportanceHelicobacter pylori established chronic infection in half of the worlds population. This infection initiates during childhood, and leads to later life gastric diseases including gastritis, peptic ulcers, and gastric cancer. These diseases are driven by host inflammation, with more severe inflammation leading to more severe disease. It is not fully understood how H. pylori controls inflammation. In this work, we used an H. pylori mouse infection model and characterized inflammation and colonization of wild-type H. pylori and a mutant that was known to cause elevated inflammation. We found that inflammation in the chronic stage undergoes surprising fluctuations, with related changes in Th17 numbers. H. pylori tlpA mutants caused offset inflammation dynamics, suggesting that H. pylori infection underlies this dynamism. Although there were not colonization dynamics at the time of inflammation fluctuations, there were differences between WT and tlpA mutant colonization at early time points. Our results suggest the chronic inflammation setting is more dynamic than previously thought and may be influenced by colonization properties of early infection.

microbiology↗

bioRxiv2022

FliL has a conserved function in diverse microbes to negatively modulate motor output via its N-terminal region

Bacterial surface sensing is often conferred by flagella. The flagellar motor protein FliL plays a key role in this process, but its exact role has been obscured by varying fliL mutant phenotypes. We reanalyzed results from studies on these fliL alleles and found they inadvertently compared mutants with differing length of the retained native N-terminal region, including the transmembrane helix (TM). We find that TM retention in the mutants that lack the native C-terminal domain results in loss of swimming and swarming motility, while alleles that completely lack the TM retain motility. We suggest FliL negatively regulates motor function via its N-terminal region, an observation that may relate to FliL function in mechanosensing.

microbiology↗

bioRxiv2021

The immunomodulatory 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 TlpAs 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 non-active ligands, glucosamine, acted as a chemotaxis antagonist, preventing the chemotaxis response to chemoattractant ligands and acted to block binding of ligands irrespective of whether they bound the membrane-distal or -proximal dCache_1 subdomains. In total, these results suggest TlpA senses multiple attractant ligands as well as antagonist ones, an emerging theme in chemotaxis systems. ImportanceNumerous 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 bacteria 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 for regulating chemotactic responses by antagonistic chemoreceptor ligands, which is an emerging theme among other chemotactic systems.

microbiology↗

bioRxiv2020

Gastric metabolomics analysis supports H. pyloris catabolism of organic and amino acids 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. It is not known, however, whether the host-derived metabolites differ between the two main regions, the corpus and antrum. We thus characterized the metabolomes of mouse gastric corpus and antrum organoids and tissue. The secreted organoid metabolites differed significantly between the corpus and antrum in only seven metabolites: lactic acid, malic acid, phosphoethanolamine, alanine, uridine, glycerol, and isoleucine, with only lactic acid and phosphoethanolamine exceeding two-fold differences. Multiple chemicals were depleted upon H. pylori infection including urea, cholesterol, glutamine, fumaric acid, lactic acid, citric acid, malic acid, and multiple non-essential amino acids. Many are known H. pylori nutrients and did not differ between the two regions. When tissue was examined, there was little overlap with the organoid metabolites. The two tissue regions were distinct, however, including in antrum-elevated 5-methoxytryptamine, lactic acid, and caprylic acid, and corpus-elevated phospholipid products. Over an 8 month infection time course, the corpus and antrum remained distinct. The antrum displayed no significant changes, in contrast to the corpus which exhibited metabolite changes indicating stress, tissue damage, and depletion of key nutrients such as glutamine and fructose-6-phosphate. Overall, our results suggest the H. pylori preferentially uses carboxylic acids and amino acids in complex environments, and these are found in both the corpus and antrum.

microbiology↗

bioRxiv2018

The Helicobacter pylori biofilm involves a multi-gene stress-biased response including a structural role for flagella

Helicobacter pylori has an impressive ability to persist chronically in the human stomach. Similar characteristics are associated with biofilm formation in other bacteria. The H. pylori biofilm process, however, is poorly understood. To gain insight into this mode of growth, we carried out comparative transcriptomic analysis between H. pylori biofilm and planktonic cells, using the mouse colonizing strain SS1. Optimal biofilm formation was obtained with low serum and three-day growth, conditions which caused both biofilm and planktonic cells to be [~]80% coccoid. RNA-seq analysis found that 8.18% of genes were differentially expressed between biofilm and planktonic cell transcriptomes. Biofilm-downregulated genes included those involved in metabolism and translation, suggesting these cells have low metabolic activity. Biofilm-upregulated genes included those whose products were predicted to be at the cell envelope, involved in regulating a stress response, and surprisingly, genes related to formation of the flagellar apparatus. Scanning electron microscopy visualized flagella that appeared to be a component of the biofilm matrix, supported by the observation that an aflagellated mutant displayed a less robust biofilm with no apparent filaments. We observed flagella in the biofilm matrix of additional H. pylori strains, supporting that flagellar use is widespread. Our data thus supports a model in which H. pylori biofilm involves a multi-gene stress-biased response, and that flagella play an important role in H. pylori biofilm formation.\n\nIMPORTANCEBiofilms, communities of bacteria that are embedded in a hydrated matrix of extracellular polymeric substances, pose a substantial health risk and are key contributors to many chronic and recurrent infections. Chronicity and recalcitrant infections are also common features associated with the ulcer-causing human pathogen H. pylori. However, relatively little is known about the role of biofilms in H. pylori pathogenesis as well as the biofilm structure itself and the genes associated with this mode of growth. In the present study, we found that H. pylori biofilm cells highly expressed genes related to cell envelope, stress response and those encoding the flagellar apparatus. Flagellar filaments were seen in high abundance in the biofilm. Flagella are known to play a role in initial biofilm formation, but typically are downregulated after that state. H. pylori instead appears to have co-opted these structures for non-motility roles, including a role building a robust biofilm.

microbiology↗

bioRxiv2017

Chemotaxis allows bacteria to overcome host-generated reactive oxygen species that constrain gland colonization

The epithelial layer of the gastrointestinal tract contains invaginations, called glands or crypts, which are colonized by symbiotic and pathogenic microorganisms and may function as designated niches for certain species. Factors that control gland colonization are poorly understood, but bacterial chemotaxis aids occupation of these sites. We report here that a Helicobacter pylori cytoplasmic chemoreceptor, TlpD, is required for gland colonization in the stomach. tlpD mutants demonstrate gland colonization defects characterized by a reduction in the percent of glands colonized, but not in number of bacteria per gland. Consistent with TlpDs reported role in reactive oxygen species (ROS) avoidance, tlpD mutants showed hallmarks of exposure to large amounts of ROS. To assess the role of host-generated ROS in TlpD-dependent gland colonization, we utilized mice that lack either the ability to generate epithelial hydrogen peroxide or immune cell superoxide. tlpD gland colonization defects were rescued to wild-type H. pylori levels in both of these mutants. These results suggest that multiple types of innate immune generated ROS production limit gland colonization and that bacteria have evolved specific mechanisms to migrate through this gauntlet to establish in the glands.\n\nClassification: Biological sciences; microbiology\n\nSignificance statementMicrobial colonization of the gastrointestinal tract occurs at distinct sites within the tissue including glandular structures found in the stomach and intestine. Multiple lines of evidence suggest that glands supply niches that promote chronic microbial colonization, a process that is critical for symbiotic and pathogenic bacteria to maintain themselves. In this report, we show that host-produced reactive oxygen species (ROS) constrain gland colonization by the gastric pathogen Helicobacter pylori. A bacterial cytoplasmic chemoreceptor, TlpD, allows H. pylori to avoid ROS and enhances H. pyloris ability to colonize a broad swath of glands. We propose that hosts limit gland access and spread by producing ROS, and bacteria counter with chemotactic responses that allow navigation through this gauntlet.

microbiology↗