Ohad Gal-Mor

Pathogenicity islands: a molecular toolbox for bacterial virulence.

Pathogenicity islands (PAIs) are distinct genetic elements on the chromosomes of a large number of bacterial pathogens. PAIs encode various virulence factors and are normally absent from non‐pathogenic strains of the same or closely related species. PAIs are considered to be a subclass of genomic islands that are acquired by horizontal gene transfer via transduction, conjugation and transformation, and provide ‘quantum leaps’ in microbial evolution. Data based on numerous sequenced bacterial genomes demonstrate that PAIs are present in a wide range of both Gram‐positive and Gram‐negative bacterial pathogens of humans, animals and plants. Recent research focused on PAIs has not only led to the identification of many novel virulence factors used by these species during infection of their respective hosts, but also dramatically changed our way of thinking about the evolution of bacterial virulence.

SseL Is a Salmonella-Specific Translocated Effector Integrated into the SsrB-Controlled Salmonella Pathogenicity Island 2 Type III Secretion System

ABSTRACT Bacterial pathogens use horizontal gene transfer to acquire virulence factors that influence host colonization, alter virulence traits, and ultimately shape the outcome of disease following infection. One hallmark of the host-pathogen interaction is the prokaryotic type III secretion system that translocates virulence factors into host cells during infection. Salmonella enterica possesses two type III secretion systems that are utilized during host colonization and intracellular replication. Salmonella pathogenicity island 2 (SPI2) is a genomic island containing approximately 30 contiguous genes required to assemble a functional secretion system including the two-component regulatory system called SsrA-SsrB that positively regulates transcription of the secretion apparatus. We used transcriptional profiling with DNA microarrays to search for genes that coregulate with the SPI2 type III secretion machinery in an SsrB-dependent manner. Here we report the identification of a Salmonella-specific translocated effector called SseL that is required for full virulence during murine typhoid-like disease. Analysis of infected macrophages using fluorescence-activated cell sorting revealed that sseL is induced inside cells and requires SsrB for expression. SseL is retained predominantly in the cytoplasm of infected cells following translocation by the type III system encoded in SPI2. Animal infection experiments with sseL mutant bacteria indicate that integration of SseL into the SsrB response regulatory system contributes to systemic virulence of this pathogen.

SseK1 and SseK2 are novel translocated proteins of Salmonella enterica serovar Typhimurium

ABSTRACT Salmonella enterica is a gram-negative, facultative intracellular pathogen that causes disease symptoms ranging from gastroenteritis to typhoid fever. A key virulence strategy is the translocation of bacterial effector proteins into the host cell, mediated by the type III secretion systems (TTSSs) encoded in Salmonella pathogenicity island 1 (SPI-1) and SPI-2. In S. enterica serovar Typhimurium LT2, we identified the protein products of STM4157 and STM2137 as novel candidate secreted proteins by comparison to known secreted proteins from enterohemorrhagic Escherichia coli and Citrobacter rodentium. The STM4157 and STM2137 proteins, which we have designated SseK1 and SseK2, respectively, are 61% identical at the amino acid level and differ mainly in their N termini. Western analysis showed that in vitro accumulation and secretion of these proteins in serovar Typhimurium were affected by mutations in the two-component systems SsrA/B and PhoP/Q, which are key mediators of intracellular growth and survival. SPI-2 TTSS-dependent translocation of recombinant SseK1::Cya was evident at 9 h postinfection of epithelial cells, while translocation of SseK2::Cya was not detected until 21 h. Remarkably, the translocation signal for SseK1 was contained within the N-terminal 32 amino acids. Fractionation of infected epithelial cells revealed that following translocation SseK1 localizes to the host cytosol, which is unusual among the currently known Salmonella effectors. Phenotypic analysis of ΔsseK1, ΔsseK2, and ΔsseK1/ΔsseK2 mutants provided evidence for a role that was not critical during systemic infection. In summary, this work demonstrates that SseK1 and SseK2 are novel translocated proteins of serovar Typhimurium.

A novel secretion pathway of Salmonella enterica acts as an antivirulence modulator during salmonellosis.

Salmonella spp. are Gram-negative enteropathogenic bacteria that infect a variety of vertebrate hosts. Like any other living organism, protein secretion is a fundamental process essential for various aspects of Salmonella biology. Herein we report the identification and characterization of a horizontally acquired, autonomous and previously unreported secretion pathway. In Salmonella enterica serovar Typhimurium, this novel secretion pathway is encoded by STM1669 and STM1668, designated zirT and zirS, respectively. We show that ZirT is localized to the bacterial outer membrane, expected to adopt a compact β-barrel conformation, and functions as a translocator for ZirS. ZirS is an exoprotein, which is secreted into the extracellular environment in a ZirT-dependent manner. The ZirTS secretion pathway was found to share several important features with two-partner secretion (TPS) systems and members of the intimin/invasin family of adhesions. We show that zirTS expression is affected by zinc; and that in vivo, induction of zirT occurs distinctively in Salmonella colonizing the small intestine, but not in systemic sites. Additionally, strong expression of zirT takes place in Salmonella shed in fecal pellets during acute and persistent infections of mice. Inactivation of ZirTS results in a hypervirulence phenotype of Salmonella during oral infection of mice. Cumulatively, these results indicate that the ZirTS pathway plays a unique role as an antivirulence modulator during systemic disease and is involved in fine-tuning a host–pathogen balance during salmonellosis.

Salmonella Phage ST64B Encodes a Member of the SseK/NleB Effector Family

Salmonella enterica is a species of bacteria that is a major cause of enteritis across the globe, while certain serovars cause typhoid, a more serious disease associated with a significant mortality rate. Type III secreted effectors are major contributors to the pathogenesis of Salmonella infections. Genes encoding effectors are acquired via horizontal gene transfer, and a subset are encoded within active phage lysogens. Because the acquisition of effectors is in flux, the complement of effectors possessed by various Salmonella strains frequently differs. By comparing the genome sequences of S. enterica serovar Typhimurium strain SL1344 with LT2, we identified a gene with significant similarity to SseK/NleB type III secreted effector proteins within a phage ST64B lysogen that is absent from LT2. We have named this gene sseK3. SseK3 was co-regulated with the SPI-2 type III secretion system in vitro and inside host cells, and was also injected into infected host cells. While no role for SseK3 in virulence could be identified, a role for the other family members in murine typhoid was found. SseK3 and other phage-encoded effectors were found to have a significant but sparse distribution in the available Salmonella genome sequences, indicating the potential for more uncharacterised effectors to be present in less studied serovars. These phage-encoded effectors may be principle subjects of contemporary selective processes shaping Salmonella-host interactions.

A unique megaplasmid contributes to stress tolerance and pathogenicity of an emergent Salmonella enterica serovar Infantis strain

Of all known Salmonella enterica serovars, S. Infantis is one of the most commonly isolated and has been recently emerging worldwide. To understand the recent emergence of S. Infantis in Israel, we performed extensive comparative analyses between pre-emergent and the clonal emergent S. Infantis populations. We demonstrate the fixation of adaptive mutations in the DNA gyrase (gyrA) and nitroreductase (nfsA) genes, conferring resistance to quinolones and nitrofurans, respectively, and the carriage of an emergent-specific plasmid, designated pESI. This self-transferred episome is a mosaic megaplasmid (∼280u2009kb), which increases bacterial tolerance to environmental mercury (mer operon) and oxidative stress, and provides further resistance to tetracycline, sulfamethoxazole and trimethoprim, most likely due to the presence of tetRA, sulI and dfrA genes respectively. Moreover, pESI carries the yersiniabactin siderophore system and two novel chaperone-usher fimbriae. In vitro studies established that pESI conjugation into a plasmidless S. Infantis strain results in superior biofilm formation, adhesion and invasion into avian and mammalian host cells. In vivo mouse infections demonstrated higher pathogenicity and increased intestinal inflammation caused by an S. Infantis strain harboring pESI compared with the plasmidless parental strain. Our results indicate that the presence of pESI that was found only in the emergent population of S. Infantis in Israel contributes significantly to antimicrobials tolerance and pathogenicity of its carrier. It is highly likely that pESI plays a key role in the successful spread of the emergent clone that replaced the local S. Infantis community in the short time of only 2-3 years.

The Salmonella enterica PhoP directly activates the horizontally acquired SPI-2 gene sseL and is functionally different from a S. bongori ortholog.

To establish a successful infection within the host, a pathogen must closely regulate multiple virulence traits to ensure their accurate temporal and spatial expression. As a highly adapted intracellular pathogen, Salmonella enterica has acquired during its evolution various virulence genes via numerous lateral transfer events, including the acquisition of the Salmonella Pathogenicity Island 2 (SPI-2) and its associated effectors. Beneficial use of horizontally acquired genes requires that their expression is effectively coordinated with the already existing virulence programs and the regulatory set-up in the bacterium. As an example for such a mechanism, we show here that the ancestral PhoPQ system of Salmonella enterica is able to regulate directly the SPI-2 effector gene sseL (encoding a secreted deubiquitinase) in an SsrB-independent manner and that PhoP plays a part in a feed-forward regulatory loop, which fine-tunes the cellular level of SseL. Additionally, we demonstrate the presence of conserved cis regulatory elements in the promoter region of sseL and show direct binding of purified PhoP to this region. Interestingly, in contrast to the S. enterica PhoP, an ortholog regulator from a S. bongori SARC 12 strain was found to be impaired in promoting transcription of sseL and other genes from the PhoP regulon. These findings have led to the identification of a previously uncharacterized residue in the DNA-binding domain of PhoP, which is required for the transcriptional activation of PhoP regulated genes in Salmonella spp. Collectively our data demonstrate an interesting interface between the acquired SsrB regulon and the ancestral PhoPQ regulatory circuit, provide novel insights into the function of PhoP, and highlight a mechanism of regulatory integration of horizontally acquired genes into the virulence network of Salmonella enterica.

Pathogens on aspirin: promising research and therapeutic applications

An aspirin-modulated gene expression control circuit provides a powerful method to regulate expression of bacterial genes inside an infected host. This should provide a safe and easy way to study host-pathogen interactions, and may have direct therapeutic applications.

Sink traps as the source of transmission of OXA-48–producing Serratia marcescens in an intensive care unit

BACKGROUNDnCarbapenemase-producing Enterobacteriaceae (CPE) outbreaks are mostly attributed to patient-to-patient transmission via healthcare workers.nnnOBJECTIVEnWe describe successful containment of a prolonged OXA-48-producing S. marcescens outbreak after recognizing the sink traps as the source of transmission.nnnMETHODSnThe Sheba Medical Center intensive care unit (ICU), contains 16 single-bed, semi-closed rooms. Active CPE surveillance includes twice-weekly rectal screening of all patients. A case was defined as a patient detected with OXA-48 CPE >72 hours after admission. A root-cause analysis was used to investigate the outbreak. All samples were inoculated on chrom-agar CRE, and carbapenemase genes were detected using commercial molecular Xpert-Carba-R. Environmental and patient S. marcescens isolates were characterized using PFGE.nnnRESULTSnFrom January 2016 to May 2017, 32 OXA-48 CPE cases were detected, and 81% of these were S. marcescens. A single clone was the cause of all but the first 2 cases. The common factor in all cases was the use of relatively large amounts of tap water. The outbreak clone was detected in 2 sink outlets and 16 sink traps. In addition to routine strict infection control measures, measures taken to contain the outbreak included (1) various sink decontamination efforts, which eliminated the bacteria from the sink drains only temporarily and (2) educational intervention that engaged the ICU team and lead to high adherence to sink-contamination prevention guidelines. No additional cases were detected for 12 months.nnnCONCLUSIONSnDespite persistence of the outbreak clones in the environmental reservoir for 1 year, the outbreak was rapidly and successfully contained. Addressing sink traps as hidden reservoirs played a major role in the intervention.

Fever-like temperature is a virulence regulatory cue controlling the motility and host cell entry of typhoidal Salmonella

........................................................................................................................... for presenting the oral talk entitled: ........................................................................................................................... ...........................................................................................................................T mucosal surfaces are important sites of entry for a majority of microorganism, and viruses in particular. Equine Herpesvirus type 1 (EHV-1) is an example of an invasive virus of the airway mucosa. An essential prerequisite for an effective host attack of the virus is to breach the epithelial cell layer and the underlying Basement Membrane (BM) barrier. In our research, nasal mucosa explants were inoculated with EHV-1 and then double immunofluorescence staining was performed to detect viral antigen positive cells as well as integrin alpha 6, laminin, collagen IV and collagen VII. The breadth of these extracellular matrix proteins was measured in Regions Of Interest (ROI) at a magnification of 200X. ROI were defined beneath non-infected and infected regions. In infected regions, the percentage of ROI were significantly decreased for integrin alpha 6 after 24 hours and 48 hours of inoculation. However, infection did not alter the percentages for laminin and collagen IV. For collagen VII, an increase in the percentage could be observed underneath EHV-1-infected plaques only at 48 hours of inoculation. In conclusion, the results revealed a substantial impact of EHV-1 infection on integrin alpha 6 and collagen VII, two important components of the extracellular matrix, which are normally associated with the basement membrane and may play a role in virus penetration to underlying tissues.T study was aimed to evaluate the effect of Milbond (HSCAS) on aflatoxin M1 in artificially contaminated cow’s milk. Chemisorption compounds used in this experiment were MIlBond, hydrated sodium calcium aluminosilicate (HSCAS). Raw cow milk were artificially exposed to aflatoxin M1 in a concentration of 100 ppb) with addition of Nilbond at 0.5, 1, 2 and 3 % at room temperature for 30 minutes. Aflatoxin M1 was decreased more than 95% by HSCAS at 2%. Milk composition consist of protein, fat, lactose, solid non fat and total solid were affected by addition of some adsorbents were not significantly affected (p 0.05). Tthis method did not involve degrading the toxin, milk may be free from toxin degradation products and is safe for consumption. In addition, the added material may be easily separated from milk after the substance adsorbs the toxin. Thus, this method should be developed by further researches for determining effects of these compounds on functional properties of milk. The ability of hydrated sodium calcium aluminosilicate to prevent or reduce the level of aflatoxin MI residues in milk is critically needed. This finding has important implications, because milk is ultimately consumed by humans and animals, and the reduction of aflatoxin contamination in the milk could have an important impact on their health.The presence of viable bacteria in the blood is commonly known as bacteremia. It can be a very localized and transient event with no consequences but for the immune-suppressed or seriously wounded people. The most severe cases can develop into sepsis, septic shock and sometimes death. Faced with suspected bacteremia, a practitioner is forced to use a broad spectrum antibiotic treatment while awaiting the results of microbiological analyses of blood samples which can last for 24 hours to 72 hours. Despite numerous efforts to shorten the time required for diagnosis, in most techniques the organism identification begins only after the blood culture turns positive. Staphylococcus aureus is one of the most frequent strains causing bacteremia. For this reason, its detection is a major challenge for health issues. We propose here to carry out the microorganism identification directly from blood culture phase. To achieve this, live bacteria are detected on an antibody based biochip without any labeling. This approach relies on a simple to operate optical technique named Surface Plasmon Resonance imaging (SPRi), recently described for pathogen detection in complex samples (ground meat, milk). Biological samples are diluted in a media specifically dedicated to this application and in accordance with the recommendations for blood cultures. Then, samples are spiked with a known amount of S. aureus and loaded on the biochip. Interactions are then recorded in real time until a positive signal appears on specific antibody due to antibody-antigen recognition. In general, a few dozens of bacteria are detected in less than ten hours in human serum. We are now focusing on the methicillin-resistant strain (MRSA versus MSSA), by the identification of the PBP2a protein, which is anchored at the cell surface and therefore, is accessible to antibodies, using the recognition capability of this antibiotic resistance marker.Objective: To determine the prevalence of yeast species in the evolution of patients with breast cancer treated with antiestrogen therapy. Materials & Methods: 30 postmenopausal patients who attended the Southern OMI Medical Center were included. The following groups were formed: Group 1 patients diagnosed with Breast Ca. treated with anti estrogens for less than one year Group 2 patients diagnosed with Breast Ca. treated with anti estrogen for 1-2 years Group 3 patients diagnosed with Breast Ca. treated with anti estrogen for 2-5 years Group 4 patients diagnosed with Breast Ca., who have completed their treatment with anti estrogen. Patients were surveyed about their symptoms, periodontal indices and then oral mucosa sample were taken. Conventional Microbiological examinations for Candida species as well as the molecular biology study data were performed. Results: Microbiological findings showed that a greater variety of species of Candida were isolated from patients who used the drug during the first two years (Group 1 and 2). Only 2 species were isolated in patients who used the drug more than two years (Group 3) and those who have completed treatment (Group 4). Conclusion: The length of intake of anti estrogens influences the growth and species of Candida, having a cumulative beneficial effect on the population studied.C clariflavum is an anaerobic, cellulosome-forming thermophile, containing in its genome, a large number of cellulosomal enzymes and a complex scaffolding system. The major cohesin-dockerin interactions of the cellulosome components were characterized, and on this basis a model of diverse cellulosome assemblies was derived. Further on, we cultivated C. clariflavum on cellobiose-, microcrystalline celluloseand switchgrass-containing media, and isolated cell-free cellulosome complexes from each culture. Gel-filtration separation of the cellulosome samples revealed two major fractions, which were analyzed by label-free LC-MS/MS in order to identify the key players of the cellulosome assemblies therein. From the 13 scaffoldins present in C. clariflavum genome, 11 were identified, and a variety of enzymes from different glycoside hydrolase and carbohydrate esterase families were identified, including glycoside hydrolase families GH48, GH9, GH5, GH30, GH11 and GH10. The expression level of the cellulosomal proteins varied as a function of the carbon source used for cultivation of the bacterium. In addition, the catalytic activity of each cellulosome was examined on different cellulosic substrates, xylan and switchgrass. The cellulosome isolated from the microcrystalline cellulose-containing medium was the most active of all the cellulosomes that were tested. The results suggest that the expression of the cellulosome proteins is regulated by the type of substrate in the growth medium. Moreover, both cell-free and cell-bound cellulosome complexes were produced which can together degrade the substrate in a synergistic manner. These observations are compatible with our proposed model of cellulosome assemblies based on in-vitro cohesin-dockerin interactions studies in this bacterium.W on discovery of novel probiotic candidates are under running. Commercially, having access to a probiotic that already has industrial functionality in addition to good viability during of food processing and shelf life of product is always advantages. Few studies have been reported regarding probiotic properties of Streptococcus thermophilus strains although this species hugely used as starter culture in the production of yogurt and other dairy products. In this study, 12 isolates of S. thermophilus, that were previously isolated from home-made dairy products, were evaluated with regard to resistance to artificial gastric (pH 2.5 containing pepsin) and intestinal (pH 8.0 containing bile and pancreatin) juices, adherence ability to Caco-2 and HT29-MTX-E12 cell lines, hydrophobicity, resistance to antibiotics, and epithelial barrier function (transepithelial electrical resistance (TER) measurement). Although it has been generally assumed that S. thermophilus strains are not resistant to stresses induces in the GIT, the results of this study revealed that susceptibility of almost all of the tested strains to simulated gastric and intestine conditions was significantly lower than for probiotic control strain L. rhamnosus GG under both simulated gastric and intestinal conditions. Regarding to adherence efficiency to human gut epithelium cell lines, the results showed 7 and 6 out of 12 isolates exhibited significantly superior adherence to Caco-2 and HT29-MTX-E12 than control probiotic L. rhamnosus GG, respectively. TER measurement showed that 3 strains were able to protect Caco-2’s tight junction. Although further investigations are necessary, our results identified some of the S. thermophilus strains as probiotic candidates worth further analysis.Objective: Resistance to antibiotics by Extended Spectrum Beta-lactamases (ESBLs) producing clinically significant bacterial strains has continuously been emerging and is a great threat to therapeutics. SHV and TEM derived ESBLs producing Enterobacteriaceae have been reported throughout the world but there is a limited data available for the molecular characterization of these enzymes in Pakistan.H foot and mouth disease (HFMD) is commonly caused by a group of Enteroviruses such as Enterovirus 71(EV71) and Coxsackievirus CVA5, CVA8 and CVA 16. Coxsackieviruses generally cause mild symptoms such as high fever, rashes and vesicles in the hand, foot and mouth but EV71 can produce more severe symptoms such as brainstem encephalitis, leading to cardiopulmonary failure and death. China experienced over 2.7 million cases of HFMD infections with 384 deaths in 2014. The lack of vaccines and antiviral drugs against EV71 highlights the urgency of developing preventative and treatment agents against EV71 to prevent further fatalities. The inactivated vaccine (IV) is well advanced in development and has good clinical trial data to support the use of the vaccine. It is ready for production in China but it remains to be investigated if the immunogenicity of the IV is able to confer protection against all EV71 sub-genotypes. Although there is data to support broad protection for some genotypes/sub-genotypes at varying efficacies, more studies need to be carried out on whether the neutralizing levels induced by IV are sufficient to protect against serious HFMD infections. New developments of experimental vaccines and antivirals are presented.