Salmonella-induced Diarrhea Occurs in the Absence of IL-8 Receptor (CXCR2)-Dependent Neutrophilic Inflammation.

BACKGROUND: Gastroenteritis is the most common manifestation of nontyphoidal Salmonella enterica infections, but little is known about the pathogenesis of diarrhea in this infection METHODS: To determine whether polymorphonuclear neutrophils (PMNs) are required for diarrhea for Salmonella colitis, we infected kanamycin-pretreated interleukin 8R (IL-8R) mutant mice and controls, both with nonmutant Slc11a1 (Nramp1, ItyR). We compared the 2 mouse strains for increases in fecal water content (diarrhea) 3 days after infection, changes in expression of ion transporters in colonic epithelial cells, proliferation of epithelial cells, and severity of infection as measured by colony-forming units (CFUs). RESULTS: The IL-8R knockout mice had fewer PMNs in the colon but the other variables we measured were unaffected except for an increase in CFUs in the colon. The pathologic changes in the cecum were similar in both groups except for the lack of PMNs in the IL-8R knockout mice. There was minimal damage to the colon more distally. CONCLUSIONS: In the early stage of Salmonella colitis, PMNs are not required for diarrhea or for the decrease in expression of colonic epithelial cell apical ion transporters. They contribute to defense against infection in the cecum but not extracolonically at this stage of Salmonella colitis.

Innate immune protection against infectious diseases by pulmonary administration of a phospholipid-conjugated TLR7 ligand.

Pulmonary administration of Toll-like receptor (TLR) ligands protects hosts from inhaled pathogens. However, systemic side effects induced by TLR stimulation limit clinical development. Here, a small-molecule TLR7 ligand conjugated with phospholipid, 1V270 (also designated TMX201), was tested for innate immune activation and its ability to prevent pulmonary infection in mice. We hypothesized that phospholipid conjugation would increase internalization by immune cells and localize the compound in the lungs, thus avoiding side effects due to systemic cytokine release. Pulmonary 1V270 administration increased innate cytokines and chemokines in bronchial alveolar lavage fluids, but neither caused systemic induction of cytokines nor B cell proliferation in distant lymphoid organs. 1V270 activated pulmonary CD11c+ dendritic cells, which migrated to local lymph nodes. However, there was minimal cell infiltration into the pulmonary parenchyma. Prophylactic administration of 1V270 significantly protected mice from lethal infection with Bacillus anthracis, Venezuelan equine encephalitis virus and H1N1 influenza virus. The maximum tolerated dose of 1V270 by pulmonary administration was 75 times the effective therapeutic dose. Therefore, pulmonary 1V270 treatment can protect the host from different infectious agents by stimulating local innate immune responses while exhibiting an excellent safety profile.

Altered expression and localization of ion transporters contribute to diarrhea in mice with Salmonella-induced enteritis.

BACKGROUND & AIMS: Salmonella enterica serovar Typhimurium is an enteropathogen that causes self-limiting diarrhea in healthy individuals, but poses a significant health threat to vulnerable populations. Our understanding of the pathogenesis of Salmonella-induced diarrhea has been hampered by the lack of a suitable mouse model. After a dose of oral kanamycin, Salmonella-infected congenic BALB/c.D2(NrampG169) mice, which carry a wild-type Nramp1 gene, develop clear manifestations of diarrhea. We used this model to elucidate the pathophysiology of Salmonella-induced diarrhea. METHODS: BALB /c.D2(NrampG169) mice were treated with kanamycin and then infected with wild-type or mutant Salmonella by oral gavage. Colon tissues were isolated and Ussing chambers, quantitative polymerase chain reaction, immunoblot, and confocal microscopy analyses were used to study function and expression of ion transporters and cell proliferation. RESULTS: Studies with Ussing chambers demonstrated reduced basal and/or adenosine 3',5'-cyclic monophosphate-mediated electrogenic ion transport in infected colonic tissues, attributable to changes in chloride or sodium transport, depending on the segment studied. The effects of infection were mediated, at least in part, by effector proteins secreted by the bacterial Salmonella pathogenicity island 1- and Salmonella pathogenicity island-2-encoded virulence systems. Infected tissue showed reduced expression of the chloride-bicarbonate exchanger down-regulated in adenoma in surface colonic epithelial cells. Cystic fibrosis transmembrane conductance regulator was internalized in colonic crypt epithelial cells without a change in overall expression levels. Confocal analyses, densitometry, and quantitative polymerase chain reaction revealed that expression of epithelial sodium channel ß was reduced in distal colons of Salmonella-infected mice. The changes in transporter expression, localization, and/or function were accompanied by crypt hyperplasia in Salmonella-infected mice. CONCLUSIONS: Salmonella infection induces diarrhea by altering expression and/or function of transporters that mediate water absorption in the colon, likely reflecting the fact that epithelial cells have less time to differentiate into surface cells when proliferation rates are increased by infection.

Activated charcoal enhanced the antigen-expression and dendritic cell maturation of the vaccine using Listeria-platform.

BACKGROUND: Listeria monocytogenes (LM) has been used as a vaccine vector based upon its ability to induce a strong cell-mediated immune response. LM inactivated with γ-irradiation retains immunogenic properties and is an attractive platform for clinical use since it would have improved safety concerns compared to live vectors. Activated charcoal has been shown to enhance expression of LM proteins such as PrfA. AIM: To investigate the effect of various growth conditions supplemented with activated charcoal on recombinant antigen expression. METHODS: We prepared γ-irradiated ovalbumin-expressing LM (LM-OVA) after growth under various culture conditions. We cultured LM-OVA at various temperatures including 25°C, 37°C and 37°C with activated charcoal and compared OVA expression by western blot analysis, dendritic cells maturation and OVA-specific T cells. RESULTS: The OVA expression was highest in γ-irradiated LM-OVA grown with activated charcoal at 37°C. Compared to other growth conditions, γ-irradiated LM-OVA grown with activated charcoal at 37°C induce better DC maturation as well as production of the highest number of antigen-specific IFN γ-secreting T cells. CONCLUSION: The further study should be demonstrated the potential to alter growth conditions to enhance OVA expression resulting for vaccine vectors, thereby improving their safety and efficacy.

Microbiological analysis of nontyphoidal Salmonella strains causing distinct syndromes of bacteremia or enteritis in HIV/AIDS patients in San Diego, California.

Recurrent invasive nontyphoidal Salmonella (NTS) infection is an AIDS-defining illness that has become less common in the developed world in the era of highly active antiretroviral therapy (HAART), while it has emerged as a major public health problem in developing countries, particularly sub-Saharan Africa. We retrospectively analyzed Salmonella (NTS) infection in HIV/AIDS patients from June 2003 until December 2009 at the University of California, San Diego (UCSD), Medical Center. Bacterial isolates from all patients were tested for selected microbiological properties, including major Salmonella (NTS) virulence loci rpoS, sodCI, spvB, and sseI. Fourteen percent of all Salmonella (NTS) cases recorded at the UCSD Medical Center during this period occurred in known HIV/AIDS patients. The clinical presentations in HIV patients fell into two distinct groups, bacteremia and enteritis. There was little clinical overlap between these two syndromes. All strains were positive for the presence of the rpoS and sodCI virulence loci, and 75% of strains were positive for the presence of the spvB and sseI loci. Antibiotic susceptibility assay showed that all strains were susceptible to trimethoprim-sulfamethoxazole and ciprofloxacin. The clinical presentation did not have a clear relationship to the CD4(+) cell count. Of the bacteremic isolates, all but one isolate, drawn from a patient with substantial enteric comorbidities, had all of the virulence genes tested, but 66% of nonbacteremic, enteritis strains also contained all the tested virulence loci. In conclusion, neither patients' CD4(+) cell count nor bacterial strain properties necessarily predicted the clinical presentation of HIV/AIDS patients with Salmonella (NTS) infection, and AIDS patients can have episodes of Salmonella enteritis without dissemination.

Diarrhea and colitis in mice require the Salmonella pathogenicity island 2-encoded secretion function but not SifA or Spv effectors.

We investigated the roles of Salmonella pathogenicity island 2 (SPI-2) and two SPI-2 effectors in Salmonella colitis and diarrhea in genetically resistant BALB/c.D2(Slc11a1) congenic mice with the wild-type Nramp1 locus. Wild-type Salmonella enterica serovar Typhimurium 14028s caused a pan-colitis, and the infected mice developed frank diarrhea with a doubling of the fecal water content. An ssaV mutant caused only a 26% increase in fecal water content, without producing the pathological changes of colitis, and it did not cause weight loss over a 1-week period of observation. However, two SPI-2 effector mutants, the spvB and sifA mutants, and a double spvB sifA mutant caused diarrhea and colitis, even though the sifA mutant was sensitive to killing by bone marrow-derived macrophages from BALB/c.D2 mice and was severely impaired in extraintestinal growth but not in growth in the cecum. These results demonstrate that systemic S. enterica infection and diarrhea/colitis are distinct pathogenic processes and that only the former requires spvB and sifA.

Effects of myeloid differentiation primary response gene 88 (MyD88) activation on Helicobacter infection in vivo and induction of a Th17 response.

BACKGROUND: Helicobacter pylori is a spiral-shaped Gram-negative microaerophilic bacterium associated with a number of gastrointestinal disorders, including gastritis, peptic ulcers, and gastric cancer. Several studies have implicated a Th17 response as a key to protective immunity against Helicobacter. MATERIALS AND METHODS: Wild type (WT) and MyD88-deficient (MyD88(-/-)) mice in the C57BL/6 background were infected with H. felis for 6 and 25 weeks and colonization density and host response evaluated. Real-time PCR was used to determine the expression of cytokines and antimicrobial peptides in the gastric tissue of mice. RESULTS: mRNA expression levels of the Th17 cytokines interleukin-17A (IL-17A) and IL-22 were markedly up-regulated in WT compared with MyD88(-/-) mice both at 6 and at 25 weeks in response to infection with H. felis, indicating that induction of Th17 responses depends on MyD88 signaling. Furthermore, reduction in the expression of Th17-dependent intestinal antimicrobial peptide lipocalin-2 was linked with increased bacterial burden in the absence of MyD88 signaling. CONCLUSION: We provide evidence showing that MyD88-dependent signaling is required for the host to induce a Th17 response for the control of Helicobacter infection.

Improving reverse vaccinology with a machine learning approach.

Reverse vaccinology aims to accelerate subunit vaccine design by rapidly predicting which proteins in a pathogenic bacterial proteome are putative protective antigens. Support vector machine classification is a machine learning approach that has been applied to solve numerous classification problems in biological sciences but has not previously been incorporated into a reverse vaccinology approach. A training data set of 136 bacterial protective antigens paired with 136 non-antigens was constructed and bioinformatic tools were used to annotate this data for predicted protein features, many of which are associated with antigenicity (i.e. extracellular localization, signal peptides and B-cell epitopes). Annotation was used to train support vector machine classifiers that exhibited a maximum accuracy of 92% for discriminating protective antigens from non-antigens as assessed by a leave-tenth-out cross-validation approach. These accuracies were superior to those achieved when annotating training data with auto and cross covariance transformations of z-descriptors for hydrophobicity, molecular size and polarity, or when classification was performed using regression methods. To further validate support vector machine classifiers, they were used to rank all the proteins in six bacterial proteomes for their antigenicity. Protective antigens from the training data were significantly recalled (enriched) in the top 75 ranked proteins for all six proteomes as assessed by a Fisher's exact test (p<0.05). This paper describes a superior workflow for performing reverse vaccinology studies and provides a benchmark training data set that can be used to evaluate future methodological improvements.

The Role of the spv Genes in Salmonella Pathogenesis.

Salmonella strains cause three main types of diseases in people: gastroenteritis, enteric (typhoid) fever, and non-typhoid extra-intestinal disease with bacteremia. Genetic analysis indicates that each clinical syndrome requires distinct sets of virulence genes, and Salmonella isolates differ in their constellation of virulence traits. The spv locus is strongly associated with strains that cause non-typhoid bacteremia, but are not present in typhoid strains. The spv region contains three genes required for the virulence phenotype in mice: the positive transcriptional regulator spvR and two structural genes spvB and spvC. SpvB and SpvC are translocated into the host cell by the Salmonella pathogenicity island-2 type-three secretion system. SpvB prevents actin polymerization by ADP-ribosylation of actin monomers, while SpvC has phosphothreonine lyase activity and has been shown to inhibit MAP kinase signaling. The exact mechanisms by which SpvB and SpvC act in concert to enhance virulence are still unclear. SpvB exhibits a cytotoxic effect on host cells and is required for delayed cell death by apoptosis following intracellular infection. Strains isolated from systemic infections of immune compromised patients, particularly HIV patients, usually carry the spv locus, strongly suggesting that CD4 T cells are required to control disease due to Salmonella that are spv positive. This association is not seen with typhoid fever, indicating that the pathogenesis and immunology of typhoid have fundamental differences from the syndrome of non-typhoid bacteremia.

Penetration and activation of brain endothelium by Salmonella enterica serovar Typhimurium.

Salmonella meningitis is a serious disease of the central nervous system, common particularly in Africa. Here, we show that Salmonella enterica serovar Typhimurium is able to adhere, invade, and penetrate human brain microvascular endothelial cells (hBMECs), the single-cell layer constituting the blood-brain barrier (BBB). Cellular invasion was dependent on host actin cytoskeleton rearrangements, while expression of a functional type III secretion system was not essential. In addition, Salmonella infection activated a proinflammatory immune response targeting neutrophil signaling and recruitment. Salmonella invasion and immune activation may represent a crucial step in the penetration of the BBB and development of Salmonella meningitis.

Mucosal adjuvant activity of cholera toxin requires Th17 cells and protects against inhalation anthrax.

Cholera toxin (CT) elicits a mucosal immune response in mice when used as a vaccine adjuvant. The mechanisms by which CT exerts its adjuvant effects are incompletely understood. We show that protection against inhalation anthrax by an irradiated spore vaccine depends on CT-mediated induction of IL-17-producing CD4 Th17 cells. Furthermore, IL-17 is involved in the induction of serum and mucosal antibody responses by CT. Th17 cells induced by CT have a unique cytokine profile compared with those induced by IL-6 and TGF-beta, and their induction by CT requires cAMP-dependent secretion of IL-1beta and beta-calcitonin gene-related peptide by dendritic cells. These findings demonstrate that Th17 cells mediate mucosal adjuvant effects of CT and identify previously unexplored pathways involved in Th17 induction that could be targeted for development of unique mucosal adjuvants.

NMR-based design and evaluation of novel bidentate inhibitors of the protein tyrosine phosphatase YopH.

We describe the use of a furanyl salicyl nitroxide derivative ('spin-labeled' compound), as a paramagnetic phosphotyrosine mimetic, to carry out a second-site screening by NMR against the PTPase YopH from Yersinia pestis. Using such a fragment-based screening approach we identified several small molecules targeting YopH that bind at sites adjacent to the spin-labeled compound. These second-site fragments were subsequently used to design and synthesize bidentate YopH inhibitors with submicromolar in vitro inhibition, selectivity against the human PTPase PTP1B, and cellular activity against Y. pseudotuberculosis. These initial compounds could result useful in elucidating the structural determinants necessary for YopH inhibition and may help in the design of even more active, selective and cell permeable compounds for the development of novel therapies against Yersiniae.

Small molecule DnaK modulators targeting the beta-domain.

The molecular chaperone DnaK is essential for the survival of bacterial pathogens in the hostile environment of the host. Hence, it is in principle a promising target for drug design but for which no current inhibitors are available apart from certain antimicrobial peptides. To this end, we have screened libraries of small molecules for their ability to interact with the substrate-binding domain of DnaK. The most promising hit from the screen was synthesized and along with its analogs subjected to further assays to determine their binding affinity and ability to interfere with bacterial growth. This work resulted in the identification of a number of compounds that bind with submicromolar affinity and capable of inhibiting Yersinia pseudotuberculosis growth more effectively than the previously characterized peptides.

The use of viral load as a surrogate marker in predicting disease progression for patients with early invasive cervical cancer with integrated human papillomavirus type 16.

OBJECTIVE: The purpose of this study was to assess the effectiveness of the use of human papillomavirus type 16 (HPV16) physical status and viral load in combination to predict clinical outcome during cervical development. STUDY DESIGN: A follow-up study was monitored in association with HPV integration and viral load in 121 cervical samples with the use of multiplex quantitative polymerase chain reaction. RESULTS: A significant increase of viral load was found earlier from preinvasive to invasive groups compared with normal groups, except with clinical staging and clinical outcome. High occurrence of integrated HPV16 was observed in preinvasive (27/44 samples) and invasive cervical carcinoma (40/68 samples). Cervical progression was observed significantly in most preinvasive (18/27 samples) and invasive cases (25/40 samples) that were infected with integrated HPV. Integrated HPV16 with significant viral load can be used as a predictive marker for tumor progression in the early stage of invasive cervical carcinoma. CONCLUSION: Integrated HPV16 in combination with viral load is a predictive indicator for tumor progression in early invasive stage but not in preinvasive and advanced invasive stage.

Structure-activity relationship studies of a novel series of anthrax lethal factor inhibitors.

We report on the identification of a novel small molecule inhibitor of anthrax lethal factor using a high-throughput screening approach. Guided by molecular docking studies, we carried out structure-activity relationship (SAR) studies and evaluated activity and selectivity of most promising compounds in in vitro enzyme inhibition assays and cellular assays. Selected compounds were further analyzed for their in vitro ADME properties, which allowed us to select two compounds for further preliminary in vivo efficacy studies. The data provided represents the basis for further pharmacology and medicinal chemistry optimizations that could result in novel anti-anthrax therapies.

ClpX contributes to innate defense peptide resistance and virulence phenotypes of Bacillus anthracis.

Bacillus anthracis is a National Institute of Allergy and Infectious Diseases Category A priority pathogen and the causative agent of the deadly disease anthrax. We applied a transposon mutagenesis system to screen for novel chromosomally encoded B. anthracis virulence factors. This approach identified ClpX, the regulatory ATPase subunit of the ClpXP protease, as essential for both the hemolytic and proteolytic phenotypes surrounding colonies of B. anthracis grown on blood or casein agar media, respectively. Deletion of clpX attenuated lethality of B. anthracis Sterne in murine subcutaneous and inhalation infection models, and markedly reduced in vivo survival of the fully virulent B. anthracis Ames upon intraperitoneal challenge in guinea pigs. The extracellular proteolytic activity dependent upon ClpX function was linked to degradation of cathelicidin antimicrobial peptides, a front-line effector of innate host defense. B. anthracis lacking ClpX were rapidly killed by cathelicidin and alpha-defensin antimicrobial peptides and lysozyme in vitro. In turn, mice lacking cathelicidin proved hyper-susceptible to lethal infection with wild-type B. anthracis Sterne, confirming cathelicidin to be a critical element of innate defense against the pathogen. We conclude that ClpX is an important factor allowing B. anthracis to subvert host immune clearance mechanisms, and thus represents a novel therapeutic target for prevention or therapy of anthrax, a foremost biodefense concern.

Myeloid differentiation primary response gene 88 is required for the resolution of otitis media.

BACKGROUND: Signaling defects in the Toll-like receptor (TLR) pathway, such as interleukin-1 receptor-associated kinase 4 deficiency, highlight the prominence of TLR signaling in the defense against bacterial disease. Because myeloid differentiation primary response gene 88 (MyD88) can transduce signals from almost all TLRs, we studied its role in otitis media (OM), the most common upper respiratory tract bacterial infectious disease in young children. METHODS: The middle ears (MEs) of wild-type (WT) and MyD88(-/-) mice were inoculated with nontypeable Haemophilus influenzae (NTHi). ME infection and inflammation were monitored for 21 days after surgery. Bone marrow-derived macrophages from WT and MyD88(-/-) mice were infected with NTHi in vitro to assess their interaction with bacteria. RESULTS: In WT mice, MyD88 expression was detected in the ME stroma at baseline. MyD88(-/-) mice displayed prolonged ME mucosal thickening and delayed recruitment of neutrophils and macrophages. Although WT mice cleared NTHi within 5 days, viable NTHi were isolated for up to 21 days in MyD88(-/-) mice. The interaction between macrophages and NTHi was significantly altered in MyD88(-/-) mice. CONCLUSIONS: In this mouse model, MyD88-mediated signaling was important for clearance of infection and resolution of inflammation in acute OM due to NTHi. The role played by innate signaling in children susceptible to chronic or recurrent OM deserves further study.

Anthrax toxins inhibit neutrophil signaling pathways in brain endothelium and contribute to the pathogenesis of meningitis.

BACKGROUND: Anthrax meningitis is the main neurological complication of systemic infection with Bacillus anthracis approaching 100% mortality. The presence of bacilli in brain autopsies indicates that vegetative bacteria are able to breach the blood-brain barrier (BBB). The BBB represents not only a physical barrier but has been shown to play an active role in initiating a specific innate immune response that recruits neutrophils to the site of infection. Currently, the basic pathogenic mechanisms by which B. anthracis penetrates the BBB and causes anthrax meningitis are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Using an in vitro BBB model, we show for the first time that B. anthracis efficiently invades human brain microvascular endothelial cells (hBMEC), the single cell layer that comprises the BBB. Furthermore, transcriptional profiling of hBMEC during infection with B. anthracis revealed downregulation of 270 (87%) genes, specifically key neutrophil chemoattractants IL-8, CXCL1 (Gro alpha) and CXCL2 (Gro beta), thereby strongly contrasting hBMEC responses observed with other meningeal pathogens. Further studies using specific anthrax toxin-mutants, quantitative RT-PCR, ELISA and in vivo assays indicated that anthrax toxins actively suppress chemokine production and neutrophil recruitment during infection, allowing unrestricted proliferation and dissemination of the bacteria. Finally, mice challenged with B. anthracis Sterne, but not the toxin-deficient strain, developed meningitis. CONCLUSIONS/SIGNIFICANCE: These results suggest a significant role for anthrax toxins in thwarting the BBB innate defense response promoting penetration of bacteria into the central nervous system. Furthermore, establishment of a mouse model for anthrax meningitis will aid in our understanding of disease pathogenesis and development of more effective treatment strategies.

In vivo efficacy of a phosphodiester TLR-9 aptamer and its beneficial effect in a pulmonary anthrax infection model.

Immunostimulatory oligonucleotide (ISS-ODN) used as adjuvants are commonly modified with phosphorothioate (PS). The PS backbone prevents nuclease degradation, but confers undesired side effects, including systemic cytokine release. Previously, R10-60, a phosphodiester (PO) ISS-ODN, was structurally optimized as an intracellular Toll-like receptor-9 agonist. Here intravenous, intradermal and intranasal administration of PO R10-60 elicit local or adaptive immune responses with minimal systemic effects compared to a prototypic PS ISS-ODN in mice. Furthermore, prophylactic intranasal administration of PO R10-60 significantly delayed death in mice exposed to respiratory anthrax comparable to the PS ISS-ODN. The pattern of cytokine release suggested that early IL-1beta production might contribute to this protective effect, which was replicated with recombinant IL-1beta injections during infection. Hence, the transient effects from a PO TLR-9 agonist may be beneficial for protection in a bacterial bioterrorism attack, by delaying the onset of systemic infection without the induction of a cytokine syndrome.