Veterans Affairs Tennessee Valley Healthcare Services

Nashville, TN, United States

Veterans Affairs Tennessee Valley Healthcare Services

Nashville, TN, United States
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Guckes K.R.,Vanderbilt University | Breland E.J.,Vanderbilt University | Zhang E.W.,Vanderbilt University | Hanks S.C.,Barnard College | And 6 more authors.
Science Signaling | Year: 2017

Bacteria use two-component systems (TCSs) to react appropriately to environmental stimuli. Typical TCSs comprise a sensor histidine kinase that acts as a receptor coupled to a partner response regulator that coordinates changes in bacterial behavior, often through its activity as a transcriptional regulator. TCS interactions are typically confined to cognate pairs of histidine kinases and response regulators. We describe two distinct TCSs in uropathogenic Escherichia coli (UPEC) that interact to mediate a response to ferric iron. The PmrAB and QseBC TCSs were both required for proper transcriptional response to ferric iron. Ferric iron induced the histidine kinase PmrB to phosphotransfer to both its cognate response regulator PmrA and the noncognate response regulator QseB, leading to transcriptional responses coordinated by both regulators. Pretreatment of the UPEC strain UTI89 with ferric iron led to increased resistance to polymyxin B that required both PmrA and QseB. Similarly, pretreatment of several UPEC isolates with ferric iron increased tolerance to polymyxin B. This study defines physiologically relevant cross-talk between TCSs in a bacterial pathogen and provides a potential mechanism for antibiotic resistance of some strains of UPEC. © 2017 The Authors.


Gaddy J.A.,Veterans Affairs Tennessee Valley Healthcare Services | Gaddy J.A.,Vanderbilt University | Radin J.N.,Vanderbilt University | Radin J.N.,Urbana University | And 8 more authors.
mBio | Year: 2015

Helicobacter pylori is one of several pathogens that persist within the host despite a robust immune response. H. pylori elicits a proinflammatory response from host epithelia, resulting in the recruitment of immune cells which manifests as gastritis. Relatively little is known about how H. pylori survives antimicrobials, including calprotectin (CP), which is present during the inflammatory response. The data presented here suggest that one way H. pylori survives the nutrient sequestration by CP is through alteration of its outer membrane. CP-treated H. pylori demonstrates increased bacterial fitness in response to further coculture with CP. Moreover, CP-treated H. pylori cultures form biofilms and demonstrate decreased cell surface hydrophobicity. In response to CP, the H. pylori Lpx lipid A biosynthetic enzymes are not fully functional. The lipid A molecules observed in H. pylori cultures treated with CP indicate that the LpxF, LpxL, and LpxR enzyme functions are perturbed. Transcriptional analysis of lpxF, lpxL, and lpxR indicates that metal restriction by CP does not control this pathway through transcriptional regulation. Analyses of H. pylori lpx mutants reveal that loss of LpxF and LpxL results in increased fitness, similar to what is observed in the presence of CP; moreover, these mutants have significantly increased biofilm formation and reduced cell surface hydrophobicity. Taken together, these results demonstrate a novel mechanism of H. pylori resistance to the antimicrobial activity of CP via lipid A modification strategies and resulting biofilm formation. IMPORTANCE Helicobacter pylori evades recognition of the host’s immune system by modifying the lipid A component of lipopolysaccharide. These results demonstrate for the first time that the lipid A modification pathway is influenced by the host’s nutritional immune response. H. pylori’s exposure to the host Mn- and Zn-binding protein calprotectin perturbs the function of 3 enzymes involved in the lipid A modification pathway. Moreover, CP treatment of H. pylori, or mutants with an altered lipid A, exhibit increased bacterial fitness and increased biofilm formation. This suggests that H. pylori modifies its cell surface structure to survive under the stress imposed by the host immune response. These results provide new insights into the molecular mechanisms that influence the biofilm lifestyle and how endotoxin modification, which renders H. pylori resistant to cationic antimicrobial peptides, can be inactivated in response to sequestration of nutrient metals. © 2015 Gaddy et al.


Wilde A.D.,Vanderbilt University | Snyder D.J.,Vanderbilt University | Putnam N.E.,Vanderbilt University | Valentino M.D.,Harvard University | And 16 more authors.
PLoS Pathogens | Year: 2015

Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction. © 2015, Public Library of Science, All Rights Reserved.


Gaddy J.A.,Veterans Affairs Tennessee Valley Healthcare Services | Gaddy J.A.,Vanderbilt University | Radin J.N.,Vanderbilt University | Radin J.N.,University of Illinois at Urbana - Champaign | And 14 more authors.
PLoS Pathogens | Year: 2014

Transition metals are necessary for all forms of life including microorganisms, evidenced by the fact that 30% of all proteins are predicted to interact with a metal cofactor. Through a process termed nutritional immunity, the host actively sequesters essential nutrient metals away from invading pathogenic bacteria. Neutrophils participate in this process by producing several metal chelating proteins, including lactoferrin and calprotectin (CP). As neutrophils are an important component of the inflammatory response directed against the bacterium Helicobacter pylori, a major risk factor for gastric cancer, it was hypothesized that CP plays a role in the host response to H. pylori. Utilizing a murine model of H. pylori infection and gastric epithelial cell co-cultures, the role CP plays in modifying H. pylori -host interactions and the function of the cag Type IV Secretion System (cag T4SS) was investigated. This study indicates elevated gastric levels of CP are associated with the infiltration of neutrophils to the H. pylori-infected tissue. When infected with an H. pylori strain harboring a functional cag T4SS, calprotectin-deficient mice exhibited decreased bacterial burdens and a trend toward increased cag T4SS -dependent inflammation compared to wild-type mice. In vitro data demonstrate that culturing H. pylori with sub-inhibitory doses of CP reduces the activity of the cag T4SS and the biogenesis of cag T4SS-associated pili in a zinc-dependent fashion. Taken together, these data indicate that zinc homeostasis plays a role in regulating the proinflammatory activity of the cag T4SS. © 2014.


Dixon B.R.E.A.,Vanderbilt University | Radin J.N.,Vanderbilt University | Radin J.N.,Urbana University | Piazuelo M.B.,Vanderbilt University | And 3 more authors.
PLoS ONE | Year: 2016

Helicobacter pylori colonization of the human stomach can lead to adverse clinical outcomes including gastritis, peptic ulcers, or gastric cancer. Current data suggest that in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization. Specifically, CD4+ T cell responses impact the pathology elicited in response to H. pylori. Because gastritis is believed to be the initiating host response to more detrimental pathological outcomes, there has been a significant interest in proinflammatory T cell cytokines, including the cytokines produced by T helper 17 cells. Th17 cells produce IL-17A, IL-17F, IL-21 and IL-22. While these cytokines have been linked to inflammation, IL-17A and IL-22 are also associated with anti-microbial responses and control of bacterial colonization. The goal of this research was to determine the role of IL-22 in activation of antimicrobial responses in models of H. pylori infection using human gastric epithelial cell lines and the mouse model of H. pylori infection. Our data indicate that IL-17A and IL-22 work synergistically to induce antimicrobials and chemokines such as IL-8, components of calprotectin (CP), lipocalin (LCN) and some β-defensins in both human and primary mouse gastric epithelial cells (GEC) and gastroids. Moreover, IL-22 and IL-17Aactivated GECs were capable of inhibiting growth of H. pylori in vitro. While antimicrobials were activated by IL-17A and IL-22 in vitro, using a mouse model of H. pylori infection, the data herein indicate that IL-22 deficiency alone does not render mice more susceptible to infection, change their antimicrobial gene transcription, or significantly change their inflammatory response.


Haley K.P.,Vanderbilt University | Gaddy J.A.,Vanderbilt University | Gaddy J.A.,Veterans Affairs Tennessee Valley Healthcare Services
Gastroenterology Research and Practice | Year: 2016

Helicobacter pylori colonizes the stomachs of greater than 50% of the world's human population making it arguably one of the most successful bacterial pathogens. Chronic H. pylori colonization results in gastritis in nearly all patients; however in a subset of people, persistent infection with H. pylori is associated with an increased risk for more severe disease outcomes including B-cell lymphoma of mucosal-associated lymphoid tissue (MALT lymphoma) and invasive adenocarcinoma. Research aimed at elucidating determinants that mediate disease progression has revealed genetic differences in both humans and H. pylori which increase the risk for developing gastric cancer. Furthermore, host diet and nutrition status have been shown to influence H. pylori-associated disease outcomes. In this review we will discuss how H. pylori is able to create a replicative niche within the hostile host environment by subverting and modifying the host-generated immune response as well as successfully competing for limited nutrients such as transition metals by deploying an arsenal of metal acquisition proteins and virulence factors. Lastly, we will discuss how micronutrient availability or alterations in the gastric microbiome may exacerbate negative disease outcomes associated with H. pylori colonization. © 2016 Kathryn P. Haley and Jennifer A. Gaddy.


Haley K.P.,Vanderbilt University | Delgado A.G.,Vanderbilt University | Piazuelo M.B.,Vanderbilt University | Mortensen B.L.,Vanderbilt University | And 8 more authors.
Infection and Immunity | Year: 2015

During infectious processes, antimicrobial proteins are produced by both epithelial cells and innate immune cells. Some of these antimicrobial molecules function by targeting transition metals and sequestering these metals in a process referred to as "nutritional immunity." This chelation strategy ultimately starves invading pathogens, limiting their growth within the vertebrate host. Recent evidence suggests that these metal-binding antimicrobial molecules have the capacity to affect bacterial virulence, including toxin secretion systems. Our previous work showed that the S100A8/S100A9 heterodimer (calprotectin, or calgranulin A/B) binds zinc and represses the elaboration of the H. pylori cag type IV secretion system (T4SS). However, there are several other S100 proteins that are produced in response to infection. We hypothesized that the zinc-binding protein S100A12 (calgranulin C) is induced in response to H. pylori infection and also plays a role in controlling H. pylori growth and virulence. To test this, we analyzed gastric biopsy specimens from H. pylori-positive and -negative patients for S100A12 expression. These assays showed that S100A12 is induced in response to H. pylori infection and inhibits bacterial growth and viability in vitro by binding nutrient zinc. Furthermore, the data establish that the zinc-binding activity of the S100A12 protein represses the activity of the cag T4SS, as evidenced by the gastric cell "hummingbird" phenotype, interleukin 8 (IL-8) secretion, and CagA translocation assays. In addition, high-resolution field emission gun scanning electron microscopy (FEG-SEM) was used to demonstrate that S100A12 represses biogenesis of the cag T4SS. Together with our previous work, these data reveal that multiple S100 proteins can repress the elaboration of an oncogenic bacterial surface organelle. © 2015, American Society for Microbiology.


PubMed | Veterans Affairs Tennessee Valley Healthcare Services and Vanderbilt University
Type: Journal Article | Journal: PloS one | Year: 2016

Helicobacter pylori colonization of the human stomach can lead to adverse clinical outcomes including gastritis, peptic ulcers, or gastric cancer. Current data suggest that in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization. Specifically, CD4+ T cell responses impact the pathology elicited in response to H. pylori. Because gastritis is believed to be the initiating host response to more detrimental pathological outcomes, there has been a significant interest in pro-inflammatory T cell cytokines, including the cytokines produced by T helper 17 cells. Th17 cells produce IL-17A, IL-17F, IL-21 and IL-22. While these cytokines have been linked to inflammation, IL-17A and IL-22 are also associated with anti-microbial responses and control of bacterial colonization. The goal of this research was to determine the role of IL-22 in activation of antimicrobial responses in models of H. pylori infection using human gastric epithelial cell lines and the mouse model of H. pylori infection. Our data indicate that IL-17A and IL-22 work synergistically to induce antimicrobials and chemokines such as IL-8, components of calprotectin (CP), lipocalin (LCN) and some -defensins in both human and primary mouse gastric epithelial cells (GEC) and gastroids. Moreover, IL-22 and IL-17A-activated GECs were capable of inhibiting growth of H. pylori in vitro. While antimicrobials were activated by IL-17A and IL-22 in vitro, using a mouse model of H. pylori infection, the data herein indicate that IL-22 deficiency alone does not render mice more susceptible to infection, change their antimicrobial gene transcription, or significantly change their inflammatory response.

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