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colonizes the stomach in about half of the world's population. strains containing the pathogenicity island ( PAI) are associated with a higher risk of gastric adenocarcinoma or peptic ulcer disease than PAI-negative strains. The PAI encodes a type IV secretion system (T4SS) that mediates delivery of the CagA effector protein as well as nonprotein bacterial constituents into gastric epithelial cells. -induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and interleukin-8 (IL-8) secretion are attributed to T4SS-dependent delivery of lipopolysaccharide metabolites and peptidoglycan into host cells, and Toll-like receptor 9 (TLR9) activation is attributed to delivery of bacterial DNA. In this study, we analyzed the bacterial energetic requirements associated with these cellular alterations. Mutant strains lacking Cagα, Cagβ, or CagE (putative ATPases corresponding to VirB11, VirD4, and VirB4 in prototypical T4SSs) were capable of T4SS core complex assembly but defective in CagA translocation into host cells. Thus, the three Cag ATPases are not functionally redundant. Cagα and CagE were required for -induced NF-κB activation, IL-8 secretion, and TLR9 activation, but Cagβ was dispensable for these responses. We identified putative ATP-binding motifs (Walker-A and Walker-B) in each of the ATPases and generated mutant strains in which these motifs were altered. Each of the Walker box mutant strains exhibited properties identical to those of the corresponding deletion mutant strains. These data suggest that Cag T4SS-dependent delivery of nonprotein bacterial constituents into host cells occurs through mechanisms different from those used for recruitment and delivery of CagA into host cells.
Copyright © 2020 American Society for Microbiology.
Osteomyelitis (OM), or inflammation of bone tissue, occurs most frequently as a result of bacterial infection and severely perturbs bone structure. OM is predominantly caused by , and even with proper treatment, OM has a high rate of recurrence and chronicity. While has been shown to infect osteoblasts, it remains unclear whether osteoclasts (OCs) are also a target of intracellular infection. Here, we demonstrate the ability of to intracellularly infect and divide within OCs. OCs were differentiated from bone marrow macrophages (BMMs) by exposure to receptor activator of nuclear factor kappa-B ligand (RANKL). By utilizing an intracellular survival assay and flow cytometry, we found that at 18 h postinfection the intracellular burden of increased dramatically in cells with at least 2 days of RANKL exposure, while the bacterial burden decreased in BMMs. To further explore the signals downstream of RANKL, we manipulated factors controlling OC differentiation, NFATc1 and alternative NF-κB, and found that intracellular bacterial growth correlates with NFATc1 levels in RANKL-treated cells. Confocal and time-lapse microscopy in mature OCs showed a range of intracellular infection that correlated inversely with -phagolysosome colocalization. The propensity of OCs to become infected, paired with their diminished bactericidal capacity compared to BMMs, could promote OM progression by allowing to evade initial immune regulation and proliferate at the periphery of lesions where OCs are most abundant. The inflammation of bone tissue is called osteomyelitis, and most cases are caused by an infection with the bacterium To date, the bone-building cells, osteoblasts, have been implicated in the progression of these infections, but not much is known about how the bone-resorbing cells, osteoclasts, participate. In this study, we show that can infect osteoclasts and proliferate inside these cells, whereas bone-residing macrophages, immune cells related to osteoclasts, destroy the bacteria. These findings elucidate a unique role for osteoclasts to harbor bacteria during infection, providing a possible mechanism by which bacteria could evade destruction by the immune system.
Copyright © 2019 Krauss et al.
The translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is a membrane protein located on the outer mitochondrial membrane. Experimentally-derived structures of mouse TSPO (mTSPO) and its homologs from bacterial species have been determined by NMR spectroscopy and X-ray crystallography, respectively. These structures and ligand interactions within the TSPO binding pocket display distinct differences. Here, we leverage experimental and computational studies to derive a unified structural model of mTSPO in the presence and absence of the TSPO ligand, PK11195, and study the effects of DPC detergent micelles on the TSPO structure and ligand binding. From this work, we conclude that that the lipid-mimetic system used to solubilize mTSPO for NMR studies thermodynamically destabilizes the protein, introduces structural perturbations, and alters the characteristics of ligand binding. Furthermore, we used Rosetta to construct a unified mTSPO model that reconciles deviating features of the mammalian and bacterial TSPO. These deviating features are likely a consequence of the detergent system used for structure determination of mTSPO by NMR. The unified mTSPO model agrees with available experimental NMR data, appears to be physically realistic (i.e. thermodynamically not frustrated as judged by the Rosetta energy function), and simultaneously shares the structural features observed in sequence-conserved regions of the bacterial proteins. Finally, we identified the binding site for an imaging ligand VUIIS8310 that is currently positioned for clinical translation using NMR spectroscopy and propose a computational model of the VUIIS8310-mTSPO complex.
BACKGROUND - Accumulating data support a protective role of Helicobacter pylori against inflammatory bowel diseases (IBD), which might be mediated by strain-specific constituents, specifically cagA expression.
AIM - To perform a systematic review and meta-analysis to more clearly define the association between CagA seropositivity and IBD.
METHODS - We identified comparative studies that included sufficient detail to determine the odds or risk of IBD, Crohn's disease (CD) or ulcerative colitis (UC) amongst individuals with vs without evidence of cagA expression (eg CagA seropositivity). Estimates were pooled using a random effects model.
RESULTS - Three clinical studies met inclusion criteria. cagA expression was represented by CagA seropositivity in all studies. Compared to CagA seronegativity overall, CagA seropositivity was associated with lower odds of IBD (OR 0.31, 95% CI 0.21-0.44) and CD (OR 0.25, 95% CI 0.17-0.38), and statistically nonsignificant lower odds for UC (OR 0.68, 95% CI 0.35-1.32). Similarly, compared to H pylori non-exposed individuals, H pylori exposed, CagA seropositive individuals had lower odds of IBD (OR 0.26, 95% CI 0.16-0.41) and CD (OR 0.23, 95% CI 0.15-0.35), but not UC (OR 0.66, 0.34-1.27). However, there was no significant difference in the odds of IBD, CD or UC between H pylori exposed, CagA seronegative and H pylori non-exposed individuals.
CONCLUSION - We found evidence for a significant association between CagA seropositive H pylori exposure and reduced odds of IBD, particularly CD, but not for CagA seronegative H pylori exposure. Additional studies are needed to confirm these findings and define underlying mechanisms.
© 2019 John Wiley & Sons Ltd.
colonizes about half of humans worldwide, and its presence in the gastric mucosa is associated with an increased risk of gastric adenocarcinoma, gastric lymphoma, and peptic ulcer disease. strains carrying the pathogenicity island (PAI) are associated with increased risk of disease progression. The PAI encodes the Cag type IV secretion system (Cag), which delivers the CagA oncoprotein and other effector molecules into human gastric epithelial cells. We visualized structures of native and mutant Cag machines on the cell envelope by cryoelectron tomography. Individual cells contain multiple Cag nanomachines, each composed of a wheel-shaped outer membrane complex (OMC) with 14-fold symmetry and an inner membrane complex (IMC) with 6-fold symmetry. CagX, CagY, and CagM are required for assembly of the OMC, whereas strains lacking Cag3 and CagT produce outer membrane complexes lacking peripheral components. The IMC, which has never been visualized in detail, is configured as six tiers in cross-section view and three concentric rings surrounding a central channel in end-on view. The IMC contains three T4SS ATPases: (i) VirB4-like CagE, arranged as a hexamer of dimers at the channel entrance; (ii) a hexamer of VirB11-like Cagα, docked at the base of the CagE hexamer; and (iii) VirD4-like Cagβ and other unspecified Cag subunits, associated with the stacked CagE/Cagα complex and forming the outermost rings. The Cag and recently solved Dot/Icm system comprise new structural prototypes for the T4SS superfamily. Bacterial type IV secretion systems (T4SSs) have been phylogenetically grouped into two subfamilies. The T4ASSs, represented by the VirB/VirD4, include "minimized" machines assembled from 12 VirB- and VirD4-like subunits and compositionally larger systems such as the Cag T4BSSs encompass systems closely related in subunit composition to the Dot/Icm Here, we present structures of native and mutant Cag machines determined by cryoelectron tomography. We identify distinct outer and inner membrane complexes and, for the first time, visualize structural contributions of all three "signature" ATPases of T4SSs at the cytoplasmic entrance of the translocation channel. Despite their evolutionary divergence, the Cag aligns structurally much more closely to the Dot/Icm than an available VirB/VirD4 subcomplex. Our findings highlight the diversity of T4SSs and suggest a structural classification scheme in which T4SSs are grouped as minimized VirB/VirD4-like or larger Cag-like and Dot/Icm-like systems.
Copyright © 2019 Hu et al.
Helicobacter pylori colonizes the human stomach and contributes to the development of gastric cancer and peptic ulcer disease. H. pylori secretes a pore-forming toxin called vacuolating cytotoxin A (VacA), which contains two domains (p33 and p55) and assembles into oligomeric structures. Using single-particle cryo-electron microscopy, we have determined low-resolution structures of a VacA dodecamer and heptamer, as well as a 3.8-Å structure of the VacA hexamer. These analyses show that VacA p88 consists predominantly of a right-handed beta-helix that extends from the p55 domain into the p33 domain. We map the regions of p33 and p55 involved in hexamer assembly, model how interactions between protomers support heptamer formation, and identify surfaces of VacA that likely contact membrane. This work provides structural insights into the process of VacA oligomerization and identifies regions of VacA protomers that are predicted to contact the host cell surface during channel formation.
Copyright © 2019 Elsevier Ltd. All rights reserved.
Calprotectin (CP) inhibits bacterial viability through extracellular chelation of transition metals. However, how CP influences general metabolism remains largely unexplored. We show here that CP restricts bioavailable Zn and Fe to the pathogen Acinetobacter baumannii, inducing an extensive multi-metal perturbation of cellular physiology. Proteomics reveals severe metal starvation, and a strain lacking the candidate Zn metallochaperone ZigA possesses altered cellular abundance of multiple essential Zn-dependent enzymes and enzymes in de novo flavin biosynthesis. The ΔzigA strain exhibits decreased cellular flavin levels during metal starvation. Flavin mononucleotide provides regulation of this biosynthesis pathway, via a 3,4-dihydroxy-2-butanone 4-phosphate synthase (RibB) fusion protein, RibBX, and authentic RibB. We propose that RibBX ensures flavin sufficiency under CP-induced Fe limitation, allowing flavodoxins to substitute for Fe-ferredoxins as cell reductants. These studies elucidate adaptation to nutritional immunity and define an intersection between metallostasis and cellular metabolism in A. baumannii.
Copyright © 2019 Elsevier Ltd. All rights reserved.
The gastric bacterium causes a persistent infection that is directly responsible for gastric ulcers and gastric cancer in some patients and protective against allergic and other immunological disorders in others. The two outcomes of the -host interaction can be modeled in mice that are infected as immunocompetent adults and as neonates, respectively. Here, we have investigated the contribution of the immunomodulator VacA to -specific local and systemic immune responses in both models. We found that neonatally infected mice are colonized at higher levels than mice infected as adults and fail to generate effector T-cell responses to the bacteria; rather, T-cell responses in neonatally infected mice are skewed toward Foxp3-positive (Foxp3) regulatory T cells that are neuropilin negative and express RORγt. We found these peripherally induced regulatory T cells (pTregs) to be enriched, in a VacA-dependent manner, not only in the gastric mucosa but also in the lungs of infected mice. Pulmonary pTreg accumulation was observed in mice that have been infected neonatally with wild-type but not in mice that have been infected as adults or mice infected with a VacA null mutant. Finally, we traced VacA to gastric lamina propria myeloid cells and show that it suppressed interleukin-23 (IL-23) expression by dendritic cells and induced IL-10 and TGF-β expression in macrophages. Taken together, the results are consistent with the idea that creates a tolerogenic environment through its immunomodulator VacA, which skews T-cell responses toward Tregs, favors persistence, and affects immunity at distant sites. has coexisted with humans for at least 60.000 years and has evolved persistence strategies that allow it to evade host immunity and colonize its host for life. The VacA protein is expressed by all strains and is required for high-level persistent infection in experimental mouse models. Here, we show that VacA targets myeloid cells in the gastric mucosa to create a tolerogenic environment that facilitates regulatory T-cell differentiation, while suppressing effector T-cell priming and functionality. Tregs that are induced in the periphery during infection can be found not only in the stomach but also in the lungs of infected mice, where they are likely to affect immune responses to allergens.
Copyright © 2019 Altobelli et al.
Infection by is the primary cause of gastric adenocarcinoma. The most potent virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent α-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces -mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect pathogenicity. We show that output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged or the parental strain in which the wild-type was replaced by with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in , demonstrating that DFMO directly affects genomic stability. Deletion of abrogated the ability of DFMO to induce rearrangements directly. In conclusion, DFMO-induced oxidative stress in leads to genomic alterations and attenuates virulence.
Acinetobacter baumannii is an important nosocomial pathogen capable of causing wound infections, pneumonia, and bacteremia. During infection, A. baumannii must acquire Zn to survive and colonize the host. Vertebrates have evolved mechanisms to sequester Zn from invading pathogens by a process termed nutritional immunity. One of the most upregulated genes during Zn starvation encodes a putative cell wall-modifying enzyme which we named ZrlA. We found that inactivation of zrlA diminished growth of A. baumannii during Zn starvation. Additionally, this mutant strain displays increased cell envelope permeability, decreased membrane barrier function, and aberrant peptidoglycan muropeptide abundances. This altered envelope increases antibiotic efficacy both in vitro and in an animal model of A. baumannii pneumonia. These results establish ZrlA as a crucial link between nutrient metal uptake and cell envelope homeostasis during A. baumannii pathogenesis, which could be targeted for therapeutic development.
Copyright © 2019 Vanderbilt University Medical Center. Published by Elsevier Inc. All rights reserved.