Hamid Hossain

Intracellular Gene Expression Profile of Listeria monocytogenes

ABSTRACT Listeria monocytogenes is a gram-positive, food-borne microorganism responsible for invasive infections with a high overall mortality. L. monocytogenes is among the very few microorganisms that can induce uptake into the host cell and subsequently enter the host cell cytosol by breaching the vacuolar membrane. We infected the murine macrophage cell line P388D1 with L. monocytogenes strain EGD-e and examined the gene expression profile of L. monocytogenes inside the vacuolar and cytosolic environments of the host cell by using whole-genome microarray and mutant analyses. We found that ∼17% of the total genome was mobilized to enable adaptation for intracellular growth. Intracellularly expressed genes showed responses typical of glucose limitation within bacteria, with a decrease in the amount of mRNA encoding enzymes in the central metabolism and a temporal induction of genes involved in alternative-carbon-source utilization pathways and their regulation. Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. A total of 41 genes were species specific, being absent from the genome of the nonpathogenic Listeria innocua CLIP 11262 strain. We also detected 25 genes that were strain specific, i.e., absent from the genome of the previously sequenced L. monocytogenes F2365 serotype 4b strain, suggesting heterogeneity in the gene pool required for intracellular survival of L. monocytogenes in host cells. Overall, our study provides crucial insights into the strategy of intracellular survival and measures taken by L. monocytogenes to escape the host cell responses.

Culture-Independent Identification of Pathogenic Bacteria and Polymicrobial Infections in the Genitourinary Tract of Renal Transplant Recipients

ABSTRACT Renal transplant recipients are predisposed to urinary tract infections caused by both common uropathogens and opportunistic bacteria resulting frequently in significant polymicrobial infections. In this study, a culture-independent 16S rRNA-based approach was established to identify unusual, fastidious, or anaerobic bacteria and to investigate bacterial diversity in urinary tract specimens. Similarly sized amplicons encompassing the V6 to V8 region of the 16S rRNA were analyzed with denaturing high-performance liquid chromatography (DHPLC) (WAVE System). Artificial mixtures of single amplicons from commonly encountered uropathogenic bacteria produced distinct peak profiles whose identities were confirmed by sequencing individually collected peak products. We evaluated the application of the method on 109 urinary tract specimens from renal transplant recipients; 100% correlation was found for culture-positive specimens, and DHPLC generated peak profiles. However, for culture-negative specimens, DHPLC facilitated the detection of novel peak profiles. DNA sequencing of these individual peaks was used to identify the bacteria involved. Thus, in PCR-positive but culture-negative samples the method allowed detection of previously known uropathogens such as Corynebacterium urealyticum and Gardnerella vaginalis, but also unusual agents including Anaerococcus lactolyticus, Bacteroides vulgatus, Dialister invisus, Fusobacterium nucleatum, Lactobacillus iners, Leptotrichia amnionii, Prevotella buccalis, Prevotella ruminicola, Rahnella aquatilis, and Streptococcus intermedius were detected as single pathogens or as constituents of polymicrobial infections. The method described is reproducible and rapidly and enables both DHPLC-based profiling and sequence-based investigation of microbial communities and polymicrobial infections. A detailed understanding of infections found in recipients of renal transplants will guide antibiotic therapy regimens and provide new perspectives for decreasing the risk of graft rejection.

Temporal transcriptomic analysis of the Listeria monocytogenes EGD-e σB regulon

BackgroundThe opportunistic food-borne gram-positive pathogen Listeria monocytogenes can exist as a free-living microorganism in the environment and grow in the cytoplasm of vertebrate and invertebrate cells following infection. The general stress response, controlled by the alternative sigma factor, σB, has an important role for bacterial survival both in the environment and during infection. We used quantitative real-time PCR analysis and immuno-blot analysis to examine σB expression during growth of L. monocytogenes EGD-e. Whole genome-based transcriptional profiling was used to identify σB-dependent genes at different growth phases.ResultsWe detected 105 σB-positively regulated genes and 111 genes which appeared to be under negative control of σB and validated 36 σB-positively regulated genes in vivo using a reporter gene fusion system.ConclusionGenes comprising the σB regulon encode solute transporters, novel cell-wall proteins, universal stress proteins, transcriptional regulators and include those involved in osmoregulation, carbon metabolism, ribosome- and envelope-function, as well as virulence and niche-specific survival genes such as those involved in bile resistance and exclusion. Ten of the σB-positively regulated genes of L. monocytogenes are absent in L. innocua. A total of 75 σB-positively regulated listerial genes had homologs in B. subtilis, but only 33 have been previously described as being σB-regulated in B. subtilis even though both species share a highly conserved σB-dependent consensus sequence. A low overlap of genes may reflects adaptation of these bacteria to their respective environmental conditions.

Structural analysis of glycolipids from Borrelia burgdorferi

In this study the lipids of Borrelia burgdorferi, the causative agent of Lyme disease, were analyzed. Lipids comprise about 25-30% of the cell dry weight. The lipid fraction could be separated by HPTLC into 11 components. Staining of these components revealed two glycolipids and two phospholipids. The glycolipids represented about 50% of the total lipids and comprised only galactose as monosaccharide constituents. By means of mass spectrometric and gas chromatographic analysis both glycolipids could be identified as alpha-galactosyl-diacylglycerolipids with different fatty acid compositions. The phospholipids were identified as phosphatidylcholine and phosphatidylglycerol. Immunoassays with sera from patients with Lyme disease showed antibody reactivity only to the glycolipids, which was present in all stages of the disease. Other lipid components seemed to be non-immunogenic in Lyme disease. The glycolipids of B. burgdorferi may be, thus, considered promising candidates for diagnosis and possibly also for vaccination.

Conjugative IncFI plasmids carrying CTX-M-15 among Escherichia coli ESBL producing isolates at a University hospital in Germany

BackgroundMulti-drug-resistant, extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, constitute an emerging public-health concern. Little data on the molecular epidemiology of ESBL producing Escherichia coli is available in Germany. Here we describe the prevalence and molecular epidemiology of ESBL producing-Escherichia coli isolates at a German University hospital.MethodsWe analysed 63 non-duplicate clinical ESBL isolates obtained over an 8-month period using PCR and sequence-based ESBL allele typing, plasmid replicon typing, phylogenetic group typing. Pulsed-field gel electrophoresis (PFGE) based genotyping and plasmid profiling was performed, as well as confirmatory DNA-based hybridization assays.ResultsExamination of the 63 Escherichia coli isolates revealed an almost equal distribution among the E. coli phylogenetic groups A, B1, B2 and D. High prevalence (36/63) of the CTX-M-15 gene was observed and an analysis of PFGE-based patterns revealed the presence of this CTX-M allele in multiple clones. Resistance to cefotaxime was a transferable trait and a commonly occurring 145.5 kb conjugative IncFI plasmid was detected in 65% of E. coli carrying the CTX-M-15 allele. The rate of transferable antibiotic resistances for GM, SXT, TET, GM-SXT-TET, SXT-TET and GM-TET was 33%, 61%, 61%, 27%, 44% and 11%, respectively. The remaining strains did not have a common IncFI plasmid but harboured transferable IncFI plasmids with sizes that ranged from 97 to 242.5 kb.ConclusionOur data demonstrate the presence of IncFI plasmids within the prevailing E. coli population in a hospital setting and suggest that the dissemination of CTX-M-15 allele is associated to lateral transfer of these well-adapted, conjugative IncFI plasmids among various E. coli genotypes.

Sepsis syndrome and death in trauma patients are associated with variation in the gene encoding tumor necrosis factor.

Objective:Patients encountering severe trauma are at risk of developing sepsis syndrome and subsequent multiple organ failure. This is often associated with fatal outcome despite survival of the initial injury. We postulate that variation of the gene coding for tumor necrosis factor (TNF)-α is associated with increased occurrence of sepsis syndrome and mortality in trauma patients. Design:Prospective cohort study; validation in an external replication sample. Setting:Tertiary academic medical center. Patients:We included 159 severely traumatized patients from a single center. Serial blood samples were analyzed for serum concentrations of TNF-α and lymphotoxin-α (LTA). We genotyped nine polymorphisms in the TNF gene and tested for an association with sepsis syndrome and outcome. Genetic associations were validated in an external replication sample (n = 76). We examined the peripheral blood transcriptome in 28 patients by whole genome-based profiling and validated the results. Interventions:None. Measurements and Main Results:Carriage of the TNF rs1800629 A allele was associated with higher TNF-α serum concentrations on the first day after trauma and during follow-up (two-sided p = 5.0 × 10−5), with development of sepsis syndrome (odds ratio 7.14, two-sided p = 1.2 × 10−6; external validation sample [n = 76]: odds ratio 3.3, one-sided p = .03), and with fatal outcome (odds ratio 7.65, two-sided p = 1.9 × 10−6). Carriage of the TNF rs1800629 A allele was associated with differential expression of genes representing stronger proinflammatory and apoptotic responses compared with carriage of the wild-type allele. Conclusions:Common TNF gene variants are associated with sepsis syndrome and death after severe injury. These findings are strongly supported by functional data and may be important for developing preemptive anti-inflammatory interventions in carriers of the risk-associated allele.

Uropathogenic E. coli Induce Different Immune Response in Testicular and Peritoneal Macrophages: Implications for Testicular Immune Privilege

Infertility affects one in seven couples and ascending bacterial infections of the male genitourinary tract by Escherichia coli are an important cause of male factor infertility. Thus understanding mechanisms by which immunocompetent cells such as testicular macrophages (TM) respond to infection and how bacterial pathogens manipulate defense pathways is of importance. Whole genome expression profiling of TM and peritoneal macrophages (PM) infected with uropathogenic E. coli (UPEC) revealed major differences in regulated genes. However, a multitude of genes implicated in calcium signaling pathways was a common feature which indicated a role of calcium-dependent nuclear factor of activated T cells (NFAT) signaling. UPEC-dependent NFAT activation was confirmed in both cultured TM and in TM in an in vivo UPEC infectious rat orchitis model. Elevated expression of NFATC2-regulated anti-inflammatory cytokines was found in TM (IL-4, IL-13) and PM (IL-3, IL-4, IL-13). NFATC2 is activated by rapid influx of calcium, an activity delineated to the pore forming toxin alpha-hemolysin by bacterial mutant analysis. Alpha-hemolysin suppressed IL-6 and TNF-α cytokine release from PM and caused differential activation of MAP kinase and AP-1 signaling pathways in TM and PM leading to reciprocal expression of key pro-inflammatory cytokines in PM (IL-1α, IL-1β, IL-6 downregulated) and TM (IL-1β, IL-6 upregulated). In addition, unlike PM, LPS-treated TM were refractory to NFκB activation shown by the absence of degradation of IκBα and lack of pro-inflammatory cytokine secretion (IL-6, TNF-α). Taken together, these results suggest a mechanism to the conundrum by which TM initiate immune responses to bacteria, while maintaining testicular immune privilege with its ability to tolerate neo-autoantigens expressed on developing spermatogenic cells.

The 2001 Giessen Cohort Study on patients with prostatitis syndrome--an evaluation of inflammatory status and search for microorganisms 10 years after a first analysis.

Summary.  During the last years tremendous changes have occurred in the epidemiologic knowledge and the diagnostic process of the prostatitis syndrome. A new worldwide‐accepted classification system has become the gold standard in contemporary literature. The aim of this study was to compare the inflammatory and infectious status of men with prostatitis syndrome with results from our study cohort from 1992. A total of 168 symptomatic men (mean age 43.2 years; range 18–79) attending the Giessen prostatitis outpatient department were included. All men underwent a standard four‐glass‐test including leucocyte analysis in all specimens. A routine search for Ureaplasma urealyticum and Chlamydia trachomatis was performed. Ejaculate analysis following World Health Organization (WHO) criteria has been performed including the evaluation of increased number of peroxidase‐positive leucocytes (PPL). Men were classified according to the National Institutes of Health (NIH) prostatitis classification. The distribution of patients according to NIH criteria is as follows: NIH II (4.2%), NIH IIIA (31.5%), NIH IIIB (50.0%) and urethroprostatitis (14.3%). Chlamydial infection was present in one man (0.6%). Only two men with increased leucocytes in prostatic secretions demonstrated ≥106 million ml−1 PPL in semen. As compared with our cohort study 10 years ago, the proportion of the different subtypes of the prostatitis syndrome have remained stable. The aetiological spectrum of chronic bacterial prostatitis has not changed whereas, in contrast, the prevalence of C. trachomatis now is found to be strikingly reduced. Using the WHO cutpoints for leucocytospermia the inclusion of seminal leucocytes to the diagnostic process has not influenced the distribution between inflammatory (type NIH IIIA) and noninflammatory (type NIH IIIB) chronic pelvic pain syndrome.

Cre-Mediated Stress Affects Sirtuin Expression Levels, Peroxisome Biogenesis and Metabolism, Antioxidant and Proinflammatory Signaling Pathways

Cre-mediated excision of loxP sites is widely used in mice to manipulate gene function in a tissue-specific manner. To analyze phenotypic alterations related to Cre-expression, we have used AMH-Cre-transgenic mice as a model system. Different Cre expression levels were obtained by investigation of C57BL/6J wild type as well as heterozygous and homozygous AMH-Cre-mice. Our results indicate that Cre-expression itself in Sertoli cells already has led to oxidative stress and lipid peroxidation (4-HNE lysine adducts), inducing PPARα/γ, peroxisome proliferation and alterations of peroxisome biogenesis (PEX5, PEX13 and PEX14) as well as metabolic proteins (ABCD1, ABCD3, MFP1, thiolase B, catalase). In addition to the strong catalase increase, a NRF2- and FOXO3-mediated antioxidative response (HMOX1 of the endoplasmic reticulum and mitochondrial SOD2) and a NF-κB activation were noted. TGFβ1 and proinflammatory cytokines like IL1, IL6 and TNFα were upregulated and stress-related signaling pathways were induced. Sertoli cell mRNA-microarray analysis revealed an increase of TNFR2-signaling components. 53BP1 recruitment and expression levels for DNA repair genes as well as for p53 were elevated and the ones for related sirtuin deacetylases affected (SIRT 1, 3-7) in Sertoli cells. Under chronic Cre-mediated DNA damage conditions a strong downregulation of Sirt1 was observed, suggesting that the decrease of this important coordinator between DNA repair and metabolic signaling might induce the repression release of major transcription factors regulating metabolic and cytokine-mediated stress pathways. Indeed, caspase-3 was activated and increased germ cell apoptosis was observed, suggesting paracrine effects. In conclusion, the observed wide stress-induced effects and metabolic alterations suggest that it is essential to use the correct control animals (Cre/Wt) with matched Cre expression levels to differentiate between Cre-mediated and specific gene-knock out-mediated effects.

Protein Kinase C-α and Arginase I Mediate Pneumolysin-Induced Pulmonary Endothelial Hyperpermeability

Antibiotics-induced release of the pore-forming virulence factor pneumolysin (PLY) in patients with pneumococcal pneumonia results in its presence days after lungs are sterile and is a major factor responsible for the induction of permeability edema. Here we sought to identify major mechanisms mediating PLY-induced endothelial dysfunction. We evaluated PLY-induced endothelial hyperpermeability in human lung microvascular endothelial cells (HL-MVECs) and human lung pulmonary artery endothelial cells in vitro and in mice instilled intratracheally with PLY. PLY increases permeability in endothelial monolayers by reducing stable and dynamic microtubule content and modulating VE-cadherin expression. These events, dependent upon an increased calcium influx, are preceded by protein kinase C (PKC)-α activation, perturbation of the RhoA/Rac1 balance, and an increase in myosin light chain phosphorylation. At later time points, PLY treatment increases the expression and activity of arginase in HL-MVECs. Arginase inhibition abrogates and suppresses PLY-induced endothelial barrier dysfunction by restoring NO generation. Consequently, a specific PKC-α inhibitor and the TNF-derived tonoplast intrinsic protein peptide, which blunts PLY-induced PKC-α activation, are able to prevent activation of arginase in HL-MVECs and to reduce PLY-induced endothelial hyperpermeability in mice. Arginase I (AI)(+/-)/arginase II (AII)(-/-) C57BL/6 mice, displaying a significantly reduced arginase I expression in the lungs, are significantly less sensitive to PLY-induced capillary leak than their wild-type or AI(+/+)/AII(-/-) counterparts, indicating an important role for arginase I in PLY-induced endothelial hyperpermeability. These results identify PKC-α and arginase I as potential upstream and downstream therapeutic targets in PLY-induced pulmonary endothelial dysfunction.