Translocation of Campylobacter concisus from the oral cavity to the intestinal tract is increasingly recognised as a contributor to inflammatory bowel disease (IBD). The C. concisus secreted protein Csep1 has emerged as a molecular marker of C. concisus strains associated with Crohn's disease, a form of IBD. However, its structure and role in inflammation remain unknown. Here, we report the X-ray crystal structure of plasmid-encoded Csep1P that reveals a unique α-helical fold with structural similarity to Helicobacter pylori cysteine-rich proteins HcpB and HcpC. Because HcpA, another Hcp family member, is known to affect monocyte differentiation, this structural similarity led us to hypothesise that Csep1P may modulate monocyte differentiation and macrophage function. Transcriptomic analysis revealed that Csep1P induced a chemokine-dominant inflammatory state in macrophages, M1-chem. Protein-level validation in both THP-1-derived and primary human macrophages confirmed this selective chemokine response. While Csep1P alone did not upregulate proinflammatory cytokines, THP-1-derived macrophages pre-incubated with Csep1P produced a higher level of proinflammatory cytokines in response to commensal Escherichia coli, which was validated on primary human macrophages. Furthermore, silencing the delta like canonical notch ligand 4 (DLL4) gene decreased the proinflammatory response of Csep1P-mediated macrophages to E. coli. Collectively, our data demonstrate that the structurally unique Csep1P reprograms macrophage response, which provides a mechanistic link between C. concisus infection and Crohn's disease pathogenesis, and identifies Csep1P as a potential target for therapeutic intervention.

Measuring the affinity of protein-ligand interactions is crucial to understanding protein function and facilitating drug discovery. Here, we present two methods for determining protein-ligand binding affinities using a thermal shift assay (TSA) with purified protein and a single ligand concentration. One approach assumes zero heat capacity (ZHC) change over small temperature ranges. In the second approach, the unfolding equilibrium constant (UEC) is determined directly from the unfolding curves, reducing calculations.

Helicobacter pylori has evolved distinct flagellar motility to colonize the human stomach. Rotation of the H. pylori flagella is driven by one of the largest known bacterial flagellar motors. In addition to the core motor components found in Escherichia coli and Salmonella enterica, the flagellar motor in H. pylori possesses many accessories that enable the bacteria to penetrate the gastric mucus layer. Here, we utilize cryoelectron tomography with molecular genetics and biochemical approaches to characterize three accessory proteins, FlgY, PflA, and PflB, and their roles in H. pylori flagellar assembly and motility. Comparative analyses of in situ flagellar motor structures from pflA, pflB, and flgY mutants and wild-type H. pylori reveal that FlgY forms a 13-fold proximal spoke-ring around the MS-ring and that PflA and PflB form an 18-fold distal spoke-ring enclosing 18 torque-generating stator complexes. We build a pseudoatomic model of the H. pylori motor by leveraging AlphaFold-predicted structures, protein-protein interactions, and in situ motor structures. Our model suggests that the FlgY spoke-ring functions as a bearing around the rotating MS-ring and as a template for stabilizing the PflA-PflB spoke-ring, thus enabling the recruitment of 18 stator complexes for high-torque generation. Overall, our study sheds light on how this spoke-ring network between the MS-ring and stator complexes enables the unique motility of H. pylori. As these accessory proteins are conserved in the phylum Campylobacterota, our findings apply broadly to a better understanding of how polar flagella help bacteria thrive in gastric and enteric niches.

The Campylobacter genus includes many pathogenic species, with Campylobacter hepaticus primarily implicated in spotty liver disease in poultry. Chemotaxis is one of the well-established mechanisms of pathogenesis of Campylobacter. The chemoreceptor Tlp3, previously studied in C. jejuni, mediates responses to diverse ligands. Differences between the ligand-binding pockets of Tlp3s in C. hepaticus and C. jejuni may influence ligand specificity and niche adaptation. Here, we report a method for production of the ligand-binding domain of C. hepaticus Tlp3 (Ch Tlp3-LBD) in Escherichia coli inclusion bodies that yields crystallisable protein. Size-exclusion chromatography analysis showed Ch Tlp3-LBD is a monomer in solution. Ch Tlp3-LBD was crystallised using PEG 6000 and LiCl as the precipitants. The crystal lattice symmetry was P2221, with unit cell geometry of a = 82.0, b = 137.7, c = 56.1 Å, and α = β = γ = 90°. X-ray diffraction data have been acquired to 1.6 Å resolution using synchrotron radiation. Estimation of the Matthews coefficient (VM = 2.8 Å3 Da−1) and the outcome of molecular replacement suggested the asymmetric unit is composed of two protein molecules. This work lays the foundation for studies towards understanding the structural basis of ligand recognition by C. hepaticus Tlp3 and its role in pathogenesis.

Motility by means of flagella plays an important role in the persistent colonization of Helicobacter pylori in the human stomach. The H. pylori flagellar motor has a complex structure that includes a periplasmic scaffold, the components of which are still being identified. Here, we report the isolation and characterization of the soluble forms of two putative essential H. pylori motor scaffold components, proteins PflA and PflB. We developed an on-column refolding procedure, overcoming the challenge of inclusion body formation in Escherichia coli. We employed mild detergent sarkosyl to enhance protein recovery and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO)-containing buffers to achieve optimal solubility and monodispersity. In addition, we showed that PflA lacking the β-rich N-terminal domain is expressed in a soluble form, and behaves as a monodisperse monomer in solution. The methods for producing the soluble, folded forms of H. pylori PflA and PflB established in this work will facilitate future biophysical and structural studies aimed at deciphering their location and their function within the flagellar motor.

Campylobacter jejuni is a very common cause of gastroenteritis, and is frequently transmitted to humans through contaminated food products or water. Importantly, C. jejuni infections have a range of short- and long-term sequelae such as irritable bowel syndrome and Guillain Barre syndrome. C. jejuni triggers disease by employing a range of molecular strategies which enable it to colonise the gut, invade the epithelium, persist intracellularly and avoid detection by the host immune response. The objective of this review is to explore and summarise recent advances in the understanding of the C. jejuni molecular factors involved in colonisation, invasion of cells, collective quorum sensing-mediated behaviours and persistence. Understanding the mechanisms that underpin the pathogenicity of C. jejuni will enable future development of effective preventative approaches and vaccines against this pathogen.

Reactive oxygen species (ROS), including the superoxide radical anion (O2 center dot-), hydrogen peroxide (H2O2), and the hydroxyl radical (center dot HO), are inherent components of bacterial metabolism in an aerobic environment. Bacteria also encounter exogenous ROS, such as those produced by the host cells during the respiratory burst. As ROS have the capacity to damage bacterial DNA, proteins, and lipids, detoxification of ROS is critical for bacterial survival. It has been recently recognised that low-molecular-weight (LMW) thiols play a central role in this process. Here, we review the emerging role of cysteine in bacterial resistance to ROS with a link to broader elements of bacterial lifestyle closely associated with cysteine-mediated oxidative stress response, including virulence and antibiotic resistance.

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.

Helicobacter pylori encodes homologues of PilM, PilN and PilO from bacteria with Type IV pili, where these proteins form a pilus alignment complex. Inactivation of pilO changes H. pylori motility in semi-solid media, suggesting a link to the chemosensory pathways or flagellar motor. Here, we showed that mutation of the pilO or pilN gene in H. pylori strain SS1 reduced the mean linear swimming speed in liquid media, implicating PilO and PilN in the function, or regulation of, the flagellar motor. We also demonstrated that the soluble variants of H. pylori PilN and PilO share common biochemical properties with their Type IV pili counterparts which suggests their adapted function in the bacterial flagellar motor may be similar to that in the Type IV pili.

Quantification of protein-ligand interactions is crucial for understanding the protein's biological function and for drug discovery. In this study, we employed three distinct approaches for determination of protein-ligand binding affinities by a thermal shift assay using a single ligand concentration. We present the results of the comparison of the performance of the conventional curve fitting (CF) method and two newly introduced methods - assuming zero heat capacity change across small temperature ranges (ZHC) and utilizing the unfolding equilibrium constant (UEC); the latter has the advantage of reducing calculations by obtaining the unfolding equilibrium constant directly from the experimental data. Our results highlight superior performance of the ZHC and UEC methods over the conventional CF method in estimating the binding affinity, irrespective of the ligand concentration. In addition, we evaluated how the new methods can be applied to high-throughput screening for potential binders, when the enthalpy (Delta H-L) and molar heat capacity change (Delta C-PL) of ligand binding are unknown. Our results suggest that, in this scenario, using the -300 cal K-1 mol(-1) assumption for Delta C-pL and either -5 kcal mol(-1) or the average enthalpy efficiency-based estimation for Delta H-L(T) can still provide reasonable estimates of the binding affinity. Incorporating the new methods into the workflow for screening of small drug-like molecules, typically conducted using single-concentration libraries, could greatly simplify and streamline the drug discovery process.

The 17th Bacterial Locomotion and Signal Transduction (BLAST) conference took place in hybrid form in Charleston, SC, USA. This conference focused on exciting new developments in the area of bacterial signaling and motility. It showcased the work of early career researchers with 50 talks and numerous posters over 4 days. Many of the presentations highlighted collaborations occurring between multiple laboratories, and incorporated approaches including physics, modeling, biochemistry, structural biology, molecular genetics, and bacterial physiology. Key themes included an expansion of our understanding of how signaling promotes success in diverse environments; an ability to analyze signaling in complex microbial communities; new approaches to identify input signals; and technological advances that are allowing a highly refined and mechanistic view of signal transduction. With 242 attendees in person and online, the field is buzzing with fresh ideas and new directions.

The bacterial flagellar motor is a molecular nanomachine, the assembly and regulation of which requires many accessory proteins. Their identity, structure and function are often discovered through characterisation of mutants with impaired motility. Here, we demonstrate the functional association of the Helicobacter pylori peptidoglycan-associated lipoprotein (HpPal) with the flagellar motor by analysing the motility phenotype of the ∆pal mutant, and present the results of the preliminary X-ray crystallographic analysis of its globular C-terminal domain HpPal-C. Purified HpPal-C behaved as a dimer in solution. Crystals of HpPal-C were grown by the hanging drop vapour diffusion method using medium molecular weight polyethylene glycol (PEG) Smear as the precipitating agent. The crystals belong to the primitive orthorhombic space group P1 with unit cell parameters a = 50.7, b = 63.0, c = 75.1 Å. X-ray diffraction data were collected to 1.8 Å resolution on the Australian Synchrotron beamline MX2. Calculation of the Matthews coefficient (VM=2.24 Å3/Da) and molecular replacement showed that the asymmetric unit contains two protein subunits. This study is an important step towards elucidation of the non-canonical role of H. pylori Pal in the regulation, or function of, the flagellar motor.

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.

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.

OBJECTIVES Intestinal colonization by ESBL Escherichia coli and its association with community-acquired MDR infections is of great concern. This review determined the worldwide prevalence of human faecal ESBL E. coli carriage and its trend in the community over the past two decades. METHODS A systematic literature search was conducted using PubMed, EMBASE and Google Scholar to retrieve articles published between 1 January 2000 and 13 February 2020 that contained data on the prevalence of faecal carriage of ESBL E. coli among healthy individuals. A cumulative (for the whole period) meta-analysis was used to estimate the global and regional pooled prevalence rates. Articles were grouped into study periods of 3 years, and subgroup meta-analyses were undertaken to examine the global pooled prevalence over time. RESULTS Sixty-two articles covering 29 872 healthy persons were included in this meta-analysis. The cumulative (2003-18) global pooled prevalence of ESBL E. coli intestinal carriage in the community was 16.5% (95% CI 14.3%-18.7%; P  <  0.001). The pooled prevalence showed an upward trend, increasing from 2.6% (95% CI 1.6%-4.0%) in 2003-05 to 21.1% (95% CI 15.8%-27.0%) in 2015-18. Over the whole period, the highest carriage rate was observed in South-East Asia (27%; 95% CI 2.9%-51.3%), while the lowest occurred in Europe (6.0%; 95% CI 4.6%-7.5%). CONCLUSIONS Globally, an 8-fold increase in the intestinal carriage rate of ESBL E. coli in the community has occurred over the past two decades. Prevention of its spread may require new therapeutic and public health strategies.

The human gastric pathogen Helicobacter pylori relies on the uptake of host-provided nutrients for its proliferation and pathogenicity. ABC transporters that mediate import of small molecules into the cytoplasm of H. pylori employ their cognate periplasmic substrate-binding proteins (SBPs) for ligand capture in the periplasm. The genome of the mouse-adapted strain SS1 of H. pylori encodes eight ABC transporter-associated SBPs, but little is known about their specificity or structure. In this study, we demonstrated that the SBP annotated as ModA binds molybdate (MoO42-, KD = 3.8 nM) and tungstate (WO42-, KD = 7.8 nM). In addition, we showed that MetQ binds D-methionine (KD = 9.5 μM), but not L-methionine, which suggests the existence of as yet unknown pathway for L-methionine uptake. Homology modelling has led to identification of the ligand-binding residues.

In the bacterial flagellar motor, the cell-wall-anchored stator uses an electrochemical gradient across the cytoplasmic membrane to generate a turning force that is applied to the rotor connected to the flagellar filament. Existing theoretical concepts for the stator function are based on the assumption that it anchors around the rotor perimeter by binding to peptidoglycan (P). The existence of another anchoring region on the motor itself has been speculated upon, but is yet to be supported by binding studies. Due to the recent advances in electron cryotomography, evidence has emerged that polar flagellar motors contain substantial proteinaceous periplasmic structures next to the stator, without which the stator does not assemble and the motor does not function. These structures have a morphology of disks, as is the case with Vibrio spp., or a round cage, as is the case with Helicobacter pylori. It is now recognized that such additional periplasmic components are a common feature of polar flagellar motors, which sustain higher torque and greater swimming speeds compared to peritrichous bacteria such as Escherichia coli and Salmonella enterica. This review summarizes the data available on the structure, composition, and role of the periplasmic scaffold in polar bacterial flagellar motors and discusses the new paradigm for how such motors assemble and function.

With the rise of bacterial resistance to conventional antibiotics, re-purposing of Food and Drug Administration (FDA) approved drugs currently used to treat non-bacteria related diseases as new leads for antibacterial drug discovery has become an attractive alternative. Ethoxzolamide (EZA), an FDA-approved diuretic acting as a human carbonic anhydrase inhibitor, is known to kill the gastric pathogenic bacterium Helicobacter pylori in vitro via an, as yet, unknown mechanism. To date, EZA activity and resistance have been investigated for only one H. pylori strain, P12. We have now performed a susceptibility and resistance study with H. pylori strains SS1 and 26695. Mutants resistant to EZA were isolated, characterized and their genomes sequenced. Resistance-conferring mutations were confirmed by backcrossing the mutations into the parent strain. As with P12, resistance to EZA in strains SS1 and 26695 does not develop easily, since the rate of spontaneous resistance acquisition was less than 10-8. Acquisition of resistance was associated with mutations in 3 genes in strain SS1, and in 6 different genes in strain 26695, indicating that EZA targets multiple systems. All resistant isolates had mutations affecting cell wall synthesis and control of gene expression. EZA's potential for treating duodenal ulcers has already been demonstrated. Our findings suggest that EZA may be developed into a novel anti-H. pylori drug.