Project Respect: experiences of seriously ill LGBTQ+ patients and partners with their health care providers.

Discrimination against lesbian, gay, bisexual, transgender, and queer (LGBTQ+) persons in health care creates barriers to serious illness care, including patients avoiding or delaying necessary care, providers disrespecting wishes of surrogates, and adverse outcomes for patients and families. A cross-sectional mixed-methods study using an online survey was used to determine the extent to which LGBTQ+ patients and spouses, partners, and widows experienced disrespectful or inadequate care due to sexual orientation or gender identity. A total of 290 LGBTQ+ patients and partners reported high levels of disrespectful and inadequate care, including 35.2% stating their provider was insensitive to them because of their identity; 30% reporting their provider was unaware of LGBTQ+ health needs; 23.1% feeling judged; 20.7% experiencing rudeness; 20.3% stating providers did not use their correct pronouns; and 19.7% reporting their treatment decisions were disregarded. Black and Hispanic patients were 2-4 times more likely than non-Hispanic White patients to report discrimination. This study demonstrated high levels of disrespectful and inadequate care towards patients and partners due to being LGBTQ+, which was especially problematic for Black and Hispanic patients and those living in politically conservative regions. Recommendations include federal and state civil rights laws to prohibit LGBTQ+ discrimination and institutional practices to address discrimination, including cultural sensitivity training for staff.

Effects of Antimicrobial Interventions on Indicator Organisms during Beef Carcass Dressing.

ABSTRACT: Beef slaughter establishments employ many interventions to help minimize the occurrence of pathogens in their products. This study explored the effectiveness of various common interventions on microbial load using the results of the Beef-Veal Carcass Baseline Survey conducted in 2014 to 2015. The Food Safety and Inspection Service analyzed swab samples taken from 1,135 carcasses at 139 establishments. These included paired samples from post-hide removal (before evisceration) and prechill (after evisceration). Samples were tested for pathogens (Salmonella and Shiga toxin-producing Escherichia coli) and indicators (E. coli, Enterobacteriaceae, coliforms, and aerobic count [AC]). The sample size for pathogen-positive samples was small, impeding the establishment of a direct correlation between interventions and pathogens. However, we observed associations between pathogen-positive rate and log AC, indicating similar intervention effectiveness of pathogens and indicators in this study. Generally, the use of interventions reduced indicator concentrations. Each intervention produced a range of effectiveness, suggesting that how interventions are applied may be as important as which interventions are applied. The range of effectiveness for single interventions was a 0.4- to 1.9-log AC reduction; for multihurdle interventions, it ranged from 1.6- to 2.9-log AC reduction. The results of this study may be used by slaughter establishments to help identify effective intervention options for pathogen reduction.

Salmonella and Shiga Toxin-Producing Escherichia coli in Products Sampled in the Food Safety and Inspection Service Raw Pork Baseline Study.

The Food Safety and Inspection Service (FSIS) conducts microbiological baseline studies to determine national prevalence of select foodborne pathogens in federally inspected meat and poultry products and to obtain data for risk assessments. The FSIS conducted a baseline study from 1 June 2017 through 31 May 2018 to characterize and determine the prevalence of Salmonella and assess the occurrence of Shiga toxin-producing Escherichia coli (STEC) in a variety of raw pork products. In total, 4,014 samples from slaughter and processing establishments were analyzed for Salmonella; a subset of these samples (1,395) from slaughter establishments were also analyzed for STEC. Analyses determined that the national prevalence of Salmonella in raw pork products was highest in comminuted products (28.9%), followed by intact cuts (5.3%) and nonintact cuts (3.9%). Less than 1% of samples analyzed were positive for the top seven STEC. Our findings indicate there is a need for additional pathogen reduction strategies for raw pork products.

Two pKM101-encoded proteins, the pilus-tip protein TraC and Pep, assemble on the Escherichia coli cell surface as adhesins required for efficient conjugative DNA transfer.

Mobile genetic elements (MGEs) encode type IV secretion systems (T4SSs) known as conjugation machines for their transmission between bacterial cells. Conjugation machines are composed of an envelope-spanning translocation channel, and those functioning in Gram-negative species additionally elaborate an extracellular pilus to initiate donor-recipient cell contacts. We report that pKM101, a self-transmissible MGE functioning in the Enterobacteriaceae, has evolved a second target cell attachment mechanism. Two pKM101-encoded proteins, the pilus-tip adhesin TraC and a protein termed Pep, are exported to the cell surface where they interact and also form higher order complexes appearing as distinct foci or patches around the cell envelope. Surface-displayed TraC and Pep are required for an efficient conjugative transfer, 'extracellular complementation' potentially involving intercellular protein transfer, and activation of a Pseudomonas aeruginosa type VI secretion system. Both proteins are also required for bacteriophage PRD1 infection. TraC and Pep are exported across the outer membrane by a mechanism potentially involving the ß-barrel assembly machinery. The pKM101 T4SS, thus, deploys alternative routing pathways for the delivery of TraC to the pilus tip or both TraC and Pep to the cell surface. We propose that T4SS-encoded, pilus-independent attachment mechanisms maximize the probability of MGE propagation and might be widespread among this translocation superfamily.

Use of chimeric type IV secretion systems to define contributions of outer membrane subassemblies for contact-dependent translocation.

Recent studies have shown that conjugation systems of Gram-negative bacteria are composed of distinct inner and outer membrane core complexes (IMCs and OMCCs, respectively). Here, we characterized the OMCC by focusing first on a cap domain that forms a channel across the outer membrane. Strikingly, the OMCC caps of the Escherichia coli pKM101 Tra and Agrobacterium tumefaciens VirB/VirD4 systems are completely dispensable for substrate transfer, but required for formation of conjugative pili. The pKM101 OMCC cap and extended pilus also are dispensable for activation of a Pseudomonas aeruginosa type VI secretion system (T6SS). Chimeric conjugation systems composed of the IMCpKM101 joined to OMCCs from the A. tumefaciens VirB/VirD4, E. coli R388 Trw, and Bordetella pertussis Ptl systems support conjugative DNA transfer in E. coli and trigger P. aeruginosa T6SS killing, but not pilus production. The A. tumefaciens VirB/VirD4 OMCC, solved by transmission electron microscopy, adopts a cage structure similar to the pKM101 OMCC. The findings establish that OMCCs are highly structurally and functionally conserved - but also intrinsically conformationally flexible - scaffolds for translocation channels. Furthermore, the OMCC cap and a pilus tip protein coregulate pilus extension but are not required for channel assembly or function.

PrgU: a suppressor of sex pheromone toxicity in Enterococcus faecalis.

Upon sensing of the peptide pheromone cCF10, Enterococcus faecalis cells carrying pCF10 produce three surface adhesins (PrgA, PrgB or Aggregation Substance, PrgC) and the Prg/Pcf type IV secretion system and, in turn, conjugatively transfer the plasmid at high frequencies to recipient cells. Here, we report that cCF10 induction is highly toxic to cells sustaining a deletion of prgU, a small orf located immediately downstream of prgB on pCF10. Upon pheromone exposure, these cells overproduce the Prg adhesins and display impaired envelope integrity, as evidenced by antibiotic susceptibility, misplaced division septa and cell lysis. Compensatory mutations in regulatory loci controlling expression of pCF10-encoded prg/pcf genes, or constitutive PrgU overproduction, block production of the Prg adhesins and render cells insensitive to pheromone. Cells engineered to overproduce PrgB, even independently of other pCF10-encoded proteins, have severely compromised cell envelopes and strong growth defects. PrgU has an RNA-binding fold, and prgB-prgU gene pairs are widely distributed among E. faecalis isolates and other enterococcal and staphylococcal species. Together, our findings support a model in which PrgU proteins represent a novel class of RNA-binding regulators that act to mitigate toxicity accompanying overproduction of PrgB-like adhesins in E. faecalis and other clinically-important Gram-positive species.

A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain.

Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly ß-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted by H. pylori The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the ß-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the ß-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly.

Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine.

UNLABELLED: Bacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that the Escherichia coli pKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning in Agrobacterium tumefaciens, Anaplasma phagocytophilum, or Wolbachia pipientis A chimeric receptor assembled from A. tumefaciens VirD4 (VirD4At) mediated transfer of a MOBQ plasmid (pML122) and A. tumefaciens effector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4At and the TraJ/VirD4At chimera with their CTDs deleted supported pML122 transfer at wild-type levels, ΔCTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4At's CTD to the VirE2 effector, although other VirD4At domains bound this substrate in vitro We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains. IMPORTANCE: Bacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to prokaryotic and eukaryotic target cells. A hexameric ATPase, the type IV coupling protein (T4CP), functions as a substrate receptor for nearly all T4SSs. Here, we report that chimeric T4CPs mediate transfer of effector proteins through the Escherichia coli pKM101-encoded conjugation system. Studies with these repurposed conjugation systems established a role for acidic C-terminal domains of T4CPs in regulating substrate translocation. Our findings advance a mechanistic understanding of T4CP receptor activity and, further, support a model in which T4SS channels function as passive conduits for any DNA or protein substrates that successfully engage with and pass through the T4CP specificity checkpoint.

Structural organization of membrane-inserted hexamers formed by Helicobacter pylori VacA toxin.

Helicobacter pylori colonizes the human stomach and is a potential cause of peptic ulceration or gastric adenocarcinoma. H. pylori secretes a pore-forming toxin known as vacuolating cytotoxin A (VacA). The 88 kDa secreted VacA protein, composed of an N-terminal p33 domain and a C-terminal p55 domain, assembles into water-soluble oligomers. The structural organization of membrane-bound VacA has not been characterized in any detail and the role(s) of specific VacA domains in membrane binding and insertion are unclear. We show that membrane-bound VacA organizes into hexameric oligomers. Comparison of the two-dimensional averages of membrane-bound and soluble VacA hexamers generated using single particle electron microscopy reveals a structural difference in the central region of the oligomers (corresponding to the p33 domain), suggesting that membrane association triggers a structural change in the p33 domain. Analyses of the isolated p55 domain and VacA variants demonstrate that while the p55 domain can bind membranes, the p33 domain is required for membrane insertion. Surprisingly, neither VacA oligomerization nor the presence of putative transmembrane GXXXG repeats in the p33 domain is required for membrane insertion. These findings provide new insights into the process by which VacA binds and inserts into the lipid bilayer to form membrane channels.

Mechanism and Function of Type IV Secretion During Infection of the Human Host.

Bacterial pathogens employ type IV secretion systems (T4SSs) for various purposes to aid in survival and proliferation in eukaryotic hosts. One large T4SS subfamily, the conjugation systems, confers a selective advantage to the invading pathogen in clinical settings through dissemination of antibiotic resistance genes and virulence traits. Besides their intrinsic importance as principle contributors to the emergence of multiply drug-resistant "superbugs," detailed studies of these highly tractable systems have generated important new insights into the mode of action and architectures of paradigmatic T4SSs as a foundation for future efforts aimed at suppressing T4SS machine function. Over the past decade, extensive work on the second large T4SS subfamily, the effector translocators, has identified a myriad of mechanisms employed by pathogens to subvert, subdue, or bypass cellular processes and signaling pathways of the host cell. An overarching theme in the evolution of many effectors is that of molecular mimicry. These effectors carry domains similar to those of eukaryotic proteins and exert their effects through stealthy interdigitation of cellular pathways, often with the outcome not of inducing irreversible cell damage but rather of reversibly modulating cellular functions. This article summarizes the major developments for the actively studied pathogens with an emphasis on the structural and functional diversity of the T4SSs and the emerging common themes surrounding effector function in the human host.

Mechanism and structure of the bacterial type IV secretion systems.

The bacterial type IV secretion systems (T4SSs) translocate DNA and protein substrates to bacterial or eukaryotic target cells generally by a mechanism dependent on direct cell-to-cell contact. The T4SSs encompass two large subfamilies, the conjugation systems and the effector translocators. The conjugation systems mediate interbacterial DNA transfer and are responsible for the rapid dissemination of antibiotic resistance genes and virulence determinants in clinical settings. The effector translocators are used by many Gram-negative bacterial pathogens for delivery of potentially hundreds of virulence proteins to eukaryotic cells for modulation of different physiological processes during infection. Recently, there has been considerable progress in defining the structures of T4SS machine subunits and large machine subassemblies. Additionally, the nature of substrate translocation sequences and the contributions of accessory proteins to substrate docking with the translocation channel have been elucidated. A DNA translocation route through the Agrobacterium tumefaciens VirB/VirD4 system was defined, and both intracellular (DNA ligand, ATP energy) and extracellular (phage binding) signals were shown to activate type IV-dependent translocation. Finally, phylogenetic studies have shed light on the evolution and distribution of T4SSs, and complementary structure-function studies of diverse systems have identified adaptations tailored for novel functions in pathogenic settings. This review summarizes the recent progress in our understanding of the architecture and mechanism of action of these fascinating machines, with emphasis on the 'archetypal' A. tumefaciens VirB/VirD4 T4SS and related conjugation systems. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

Role of connexin 43 in Helicobacter pylori VacA-induced cell death.

Helicobacter pylori colonizes the human stomach and confers an increased risk for the development of peptic ulceration, noncardia gastric adenocarcinoma, and gastric lymphoma. A secreted H. pylori toxin, VacA, can cause multiple alterations in gastric epithelial cells, including cell death. In this study, we sought to identify host cell factors that are required for VacA-induced cell death. To do this, we analyzed gene trap and short hairpin RNA (shRNA) libraries in AZ-521 human gastric epithelial cells and selected for VacA-resistant clones. Among the VacA-resistant clones, we identified multiple gene trap library clones and an shRNA library clone with disrupted expression of connexin 43 (Cx43) (also known as gap junction protein alpha 1 [GJA1]). Further experiments with Cx43-specific shRNAs confirmed that a reduction in Cx43 expression results in resistance to VacA-induced cell death. Immunofluorescence microscopy experiments indicated that VacA did not colocalize with Cx43. We detected production of the Cx43 protein in AZ-521 cells but not in AGS, HeLa, or RK-13 cells, and correspondingly, AZ-521 cells were the most susceptible to VacA-induced cell death. When Cx43 was expressed in HeLa cells, the cells became more susceptible to VacA. These results indicate that Cx43 is a host cell constituent that contributes to VacA-induced cell death and that variation among cell types in susceptibility to VacA-induced cell death is attributable at least in part to cell type-specific differences in Cx43 production.

Flagellar localization of a Helicobacter pylori autotransporter protein.

UNLABELLED: Helicobacter pylori contains four genes that are predicted to encode proteins secreted by the autotransporter (type V) pathway. One of these, the pore-forming toxin VacA, has been studied in great detail, but thus far there has been very little investigation of three VacA-like proteins. We show here that all three VacA-like proteins are >250 kDa in mass and localized on the surface of H. pylori. The expression of the three vacA-like genes is upregulated during H. pylori colonization of the mouse stomach compared to H. pylori growth in vitro, and a wild-type H. pylori strain outcompeted each of the three corresponding isogenic mutant strains in its ability to colonize the mouse stomach. One of the VacA-like proteins localizes to a sheath that overlies the flagellar filament and bulb, and therefore, we designate it FaaA (flagella-associated autotransporter A). In comparison to a wild-type H. pylori strain, an isogenic faaA mutant strain exhibits decreased motility, decreased flagellar stability, and an increased proportion of flagella in a nonpolar site. The flagellar localization of FaaA differs markedly from the localization of other known autotransporters, and the current results reveal an important role of FaaA in flagellar localization and motility. IMPORTANCE: The pathogenesis of most bacterial infections is dependent on the actions of secreted proteins, and proteins secreted by the autotransporter pathway constitute the largest family of secreted proteins in pathogenic Gram-negative bacteria. In this study, we analyzed three autotransporter proteins (VacA-like proteins) produced by Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach and contributes to the pathogenesis of gastric cancer and peptic ulcer disease. We demonstrate that these three proteins each enhance the capacity of H. pylori to colonize the stomach. Unexpectedly, one of these proteins (FaaA) is localized to a sheath that overlies H. pylori flagella. The absence of FaaA results in decreased H. pylori motility as well as a reduction in flagellar stability and a change in flagellar localization. The atypical localization of FaaA reflects a specialized function of this autotransporter designed to optimize H. pylori colonization of the gastric niche.

Structural analysis of the oligomeric states of Helicobacter pylori VacA toxin.

Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to peptic ulceration and gastric adenocarcinoma. H. pylori secretes a pore-forming exotoxin known as vacuolating toxin (VacA). VacA contains two distinct domains, designated p33 and p55, and assembles into large "snowflake"-shaped oligomers. Thus far, no structural data are available for the p33 domain, which is essential for membrane channel formation. Using single-particle electron microscopy and the random conical tilt approach, we have determined the three-dimensional structures of six VacA oligomeric conformations at ~15-Å resolution. The p55 domain, composed primarily of ß-helical structures, localizes to the peripheral arms, while the p33 domain consists of two globular densities that localize within the center of the complexes. By fitting the VacA p55 crystal structure into the electron microscopy densities, we have mapped inter-VacA interactions that support oligomerization. In addition, we have examined VacA variants/mutants that differ from wild-type (WT) VacA in toxin activity and/or oligomeric structural features. Oligomers formed by VacA∆6-27, a mutant that fails to form membrane channels, lack an organized p33 central core. Mixed oligomers containing both WT and VacA∆6-27 subunits also lack an organized core. Oligomers formed by a VacA s2m1 chimera (which lacks cell-vacuolating activity) and VacAΔ301-328 (which retains vacuolating activity) each contain p33 central cores similar to those of WT oligomers. By providing the most detailed view of the VacA structure to date, these data offer new insights into the toxin's channel-forming component and the intermolecular interactions that underlie oligomeric assembly.

The intermediate region of Helicobacter pylori VacA is a determinant of toxin potency in a Jurkat T cell assay.

Colonization of the human stomach with Helicobacter pylori is a risk factor for peptic ulceration, noncardia gastric adenocarcinoma, and gastric lymphoma. The secreted VacA toxin is an important H. pylori virulence factor that causes multiple alterations in gastric epithelial cells and T cells. Several families of vacA alleles have been described, and H. pylori strains containing certain vacA types (s1, i1, and m1) are associated with an increased risk of gastric disease, compared to strains containing other vacA types (s2, i2, and m2). Thus far, there has been relatively little study of the role of the VacA intermediate region (i-region) in toxin activity. In this study, we compared the ability of i1 and i2 forms of VacA to cause functional alterations in Jurkat cells. To do this, we manipulated the chromosomal vacA gene in two H. pylori strains to introduce alterations in the region encoding the VacA i-region. We did not detect any differences in the capacity of i1 and i2 forms of VacA to cause vacuolation of RK13 cells. In comparison to i1 forms of VacA, i2 forms of VacA had a diminished capacity to inhibit the activation of nuclear factor of activated T cells (NFAT) and suppress interleukin-2 (IL-2) production. Correspondingly, i2 forms of VacA bound to Jurkat cells less avidly than did i1 forms of VacA. These results indicate that the VacA i-region is an important determinant of VacA effects on human T cell function.

Helicobacter pylori VacA induces programmed necrosis in gastric epithelial cells.

Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to the development of peptic ulcer disease and gastric cancer. The secreted pore-forming toxin VacA is one of the major virulence factors of H. pylori. In the current study, we show that AZ-521 human gastric epithelial cells are highly susceptible to VacA-induced cell death. Wild-type VacA causes death of these cells, whereas mutant VacA proteins defective in membrane channel formation do not. Incubation of AZ-521 cells with wild-type VacA results in cell swelling, poly(ADP-ribose) polymerase (PARP) activation, decreased intracellular ATP concentration, and lactate dehydrogenase (LDH) release. VacA-induced death of these cells is a caspase-independent process that results in cellular release of histone-binding protein high mobility group box 1 (HMGB1), a proinflammatory protein. These features are consistent with the occurrence of cell death through a programmed necrosis pathway and suggest that VacA can be included among the growing number of bacterial pore-forming toxins that induce cell death through programmed necrosis. We propose that VacA augments H. pylori-induced mucosal inflammation in the human stomach by causing programmed necrosis of gastric epithelial cells and subsequent release of proinflammatory proteins and may thereby contribute to the pathogenesis of gastric cancer and peptic ulceration.

Reconstitution of Helicobacter pylori VacA toxin from purified components.

Helicobacter pylori VacA is a pore-forming toxin that causes multiple alterations in human cells and contributes to the pathogenesis of peptic ulcer disease and gastric cancer. The toxin is secreted by H. pylori as an 88 kDa monomer (p88) consisting of two domains (p33 and p55). While an X-ray crystal structure for p55 exists and p88 oligomers have been visualized by cryo-electron microscopy, a detailed analysis of p33 has been hindered by an inability to purify this domain in an active form. In this study, we expressed and purified a recombinant form of p33 under denaturing conditions and optimized conditions for the refolding of the soluble protein. We show that refolded p33 can be added to purified p55 in trans to cause vacuolation of HeLa cells and inhibition of IL-2 production by Jurkat cells, effects identical to those produced by the p88 toxin from H. pylori. The p33 protein markedly enhances the cell binding properties of p55. Size exclusion chromatography experiments suggest that p33 and p55 assemble into a complex consistent with the size of a p88 monomer. Electron microscopy of these p33/p55 complexes reveals small rod-shaped structures that can convert to oligomeric flower-shaped structures in the presence of detergent. We propose that the oligomerization observed in these experiments mimics the process by which VacA oligomerizes when in contact with membranes of host cells.