Sangwei Lu

Recombinant Mycobacterium tuberculosis protein associated with mammalian cell entry

The ability to gain entry and resist the antimicrobial intracellular environment of mammalian cells is an essential virulence property of Mycobacterium tuberculosis. A purified recombinant protein expressed by a 1362 bp locus (mce1) in the M. tuberculosis genome promoted uptake into HeLa cells of polystyrene latex microspheres coated with the protein. N‐terminus deletion constructs of Mce1 identified a domain located between amino acid positions 106 and 163 that was needed for this cell uptake activity. Mce1 contained hydrophobic stretches at the N‐terminus predictive of a signal sequence, and colloidal gold immunoelectron microscopy indicated that the corresponding native protein is expressed on the surface of the M. tuberculosis organism. The complete M. tuberculosis genome sequence revealed that it contained four homologues of mce (mce1, mce2, mce3, mce4) and that they were all located within operons composed of genes arranged similarly at different locations in the chromosome. Recombinant Mce2, which had the highest level of identity (67%) to Mce1, was unable to promote the association of microspheres with HeLa cells. Although the exact function of Mce1 is still unknown, it appears to serve as an effector molecule expressed on the surface of M. tuberculosis that is capable of eliciting plasma membrane perturbations in non‐phagocytic mammalian cells.

Identification of Genes Associated with Survival of Salmonella enterica Serovar Enteritidis in Chicken Egg Albumen

ABSTRACT Salmonella enterica consists of over 2,000 serovars that are major causes of morbidity and mortality associated with contaminated food. Despite similarities among serovars of Salmonella enterica, many demonstrate unique host specificities, epidemiological characteristics, and clinical manifestations. One of the unique epidemiological characteristics of the serovar Enteritidis is that it is the only bacterium routinely transmitted to humans through intact chicken eggs. Therefore, Salmonella enterica serovar Enteritidis must be able to persist inside chicken eggs to be transmitted to humans, and its survival in egg is important for its transmission to the human population. The ability of Salmonella enterica serovar Enteritidis to survive in and transmit through eggs may have contributed to its drastically increased prevalence in the 1980s and 1990s. In the present study, using transposon-mediated mutagenesis, we have identified genes important for the association of Salmonella enterica serovar Enteritidis with chicken eggs. Our results indicate that genes involved in cell wall structural and functional integrity, and nucleic acid and amino acid metabolism are important for Salmonella enterica serovar Enteritidis to persist in egg albumen. Two regions unique to Salmonella enterica serovar Enteritidis were also identified, one of which enhanced the survival of a Salmonella enterica serovar Typhimurium isolate in egg albumen. The implication of our results to the serovar specificity of Salmonella enterica is also explored in the present study.

Association of Salmonella enterica serovar enteritidis yafD with resistance to chicken egg albumen.

ABSTRACT Salmonella enterica serovar Enteritidis is a major cause of food-borne diseases in industrialized countries. The incidence of S. enterica serovar Enteritidis infections has increased substantially in recent decades, and S. enterica serovar Enteritidis is now one of the leading serovars of Salmonella in the United States. A unique epidemiological characteristic of S. enterica serovar Enteritidis is its association with chicken shell eggs, since approximately 80% of all human gastrointestinal diseases can be traced to contaminated egg products. Eggs are contaminated when bacteria from reproductive tissues of infected hens are packaged into the eggs and persist inside the hostile egg albumen environment. Therefore, resistance to egg albumen is an important aspect in the transmission of S. enterica serovar Enteritidis. We identified a gene, yafD from S. enterica serovar Enteritidis, whose overexpression conferred upon S. enterica serovar Typhimurium enhanced resistance to egg albumen, while disruption of this gene in S. enterica serovar Enteritidis rendered the organism more susceptible to egg albumen. YafD is homologous to members of an exonuclease-endonuclease-phosphatase family, including some enzymes involved in DNA repair. Furthermore, we discovered that egg albumen has nuclease activities and uses both circular and linear DNA as substrates. We propose that YafD provides a survival advantage to S. enterica serovar Enteritidis in eggs by repairing DNA damage caused by egg albumen and that it may be one of the biologic determinants that contribute to the epidemiological association of S. enterica serovar Enteritidis with egg products.

The Global Regulator ArcA Controls Resistance to Reactive Nitrogen and Oxygen Intermediates in Salmonella enterica Serovar Enteritidis

ABSTRACT Salmonella enterica serovar Enteritidis is a major cause of food-borne diseases associated with consumption of shell eggs. Clinical isolates of S. enterica serovar Enteritidis exhibit a wide spectrum of virulence in mice. A highly virulent isolate (SE2472) was previously shown to be more resistant in vitro than other clinical isolates to acidified sodium nitrite (ASN), a generator of reactive nitrogen and oxygen intermediates (RNI/ROI). SE2472 is also more resistant to S-nitrosoglutathione (GSNO) and hydrogen peroxide (H2O2) than an ASN-susceptible isolate of S. enterica serovar Enteritidis (SE8743). To investigate the molecular basis for the RNI/ROI resistance of S. enterica serovar Enteritidis, we transformed a genomic DNA library of SE2472 into SE8743. A plasmid clone conferred upon SE8743 enhanced resistance to ASN, GSNO, and H2O2. The DNA insert in the clone encoded ArcA, a global regulator. An arcA mutant of SE2472 was constructed and was found to be more susceptible to GSNO and hydrogen peroxide but not more susceptible to ASN than wild-type SE2472. The susceptibility of the arcA mutant to GSNO and H2O2 was complemented by a plasmid harboring arcA. The coding sequence of the arcA gene in SE2472 and the coding sequence of the arcA gene in SE8743 were identical, suggesting that the difference in resistance to RNI/ROI maybe due to the activity of genes regulated by ArcA. No significant difference in virulence between the wild type and the arcA mutant of SE2472 was observed in mice. These observations show that arcA is essential for resistance of S. enterica serovar Enteritidis to nitrosative and oxidative stress. However, additional genetic loci may contribute to the resistance to RNI/ROI and unusually high virulence for mice of SE2472.

A Salmonella small non-coding RNA facilitates bacterial invasion and intracellular replication by modulating the expression of virulence factors.

Small non-coding RNAs (sRNAs) that act as regulators of gene expression have been identified in all kingdoms of life, including microRNA (miRNA) and small interfering RNA (siRNA) in eukaryotic cells. Numerous sRNAs identified in Salmonella are encoded by genes located at Salmonella pathogenicity islands (SPIs) that are commonly found in pathogenic strains. Whether these sRNAs are important for Salmonella pathogenesis and virulence in animals has not been reported. In this study, we provide the first direct evidence that a pathogenicity island-encoded sRNA, IsrM, is important for Salmonella invasion of epithelial cells, intracellular replication inside macrophages, and virulence and colonization in mice. IsrM RNA is expressed in vitro under conditions resembling those during infection in the gastrointestinal tract. Furthermore, IsrM is found to be differentially expressed in vivo, with higher expression in the ileum than in the spleen. IsrM targets the mRNAs coding for SopA, a SPI-1 effector, and HilE, a global regulator of the expression of SPI-1 proteins, which are major virulence factors essential for bacterial invasion. Mutations in IsrM result in disregulation of expression of HilE and SopA, as well as other SPI-1 genes whose expression is regulated by HilE. Salmonella with deletion of isrM is defective in bacteria invasion of epithelial cells and intracellular replication/survival in macrophages. Moreover, Salmonella with mutations in isrM is attenuated in killing animals and defective in growth in the ileum and spleen in mice. Our study has shown that IsrM sRNA functions as a pathogenicity island-encoded sRNA directly involved in Salmonella pathogenesis in animals. Our results also suggest that sRNAs may represent a distinct class of virulence factors that are important for bacterial infection in vivo.

Release of extracellular ATP by bacteria during growth

BackgroundAdenosine triphosphate (ATP) is used as an intracellular energy source by all living organisms. It plays a central role in the respiration and metabolism, and is the most important energy supplier in many enzymatic reactions. Its critical role as the energy storage molecule makes it extremely valuable to all cells.ResultsWe report here the detection of extracellular ATP in the cultures of a variety of bacterial species. The levels of the extracellular ATP in bacterial cultures peaked around the end of the log phase and decreased in the stationary phase of growth. Extracellular ATP levels were dependent on the cellular respiration as bacterial mutants lacking cytochrome bo oxidase displayed lower extracellular ATP levels. We have also shown that Escherichia coli (E. coli) and Salmonella actively depleted extracellular ATP and an ATP supplement in culture media enhanced the stationary survival of E. coli and Salmonella. In addition to E. coli and Salmonella the presence of the extracellular ATP was observed in a variety of bacterial species that contain human pathogens such as Acinetobacter, Pseudomonas, Klebsiella and Staphylococcus.ConclusionOur results indicate that extracellular ATP is produced by many bacterial species during growth and extracellular ATP may serve a role in the bacterial physiology.

Yeast two hybrid analyses reveal novel binary interactions between human cytomegalovirus-encoded virion proteins.

Human cytomegalovirus (HCMV) is the largest human herpesvirus and its virion contains many viral encoded proteins found in the capsid, tegument, and envelope. In this study, we carried out a yeast two-hybrid (YTH) analysis to study potential binary interactions among 56 HCMV-encoded virion proteins. We have tested more than 3,500 pairwise combinations for binary interactions in the YTH analysis, and identified 79 potential interactions that involve 37 proteins. Forty five of the 79 interactions were also identified in human cells expressing the viral proteins by co-immunoprecipitation (co-IP) experiments. To our knowledge, 58 of the 79 interactions revealed by YTH analysis, including those 24 that were also identified in co-IP experiments, have not been reported before. Novel potential interactions were found between viral capsid proteins and tegument proteins, between tegument proteins, between tegument proteins and envelope proteins, and between envelope proteins. Furthermore, both the YTH and co-IP experiments have identified 9, 7, and 5 interactions that were involved with UL25, UL24, and UL89, respectively, suggesting that these “hub” proteins may function as the organizing centers for connecting multiple virion proteins in the mature virion and for recruiting other virion proteins during virion maturation and assembly. Our study provides a framework to study potential interactions between HCMV proteins and investigate the roles of protein-protein interactions in HCMV virion formation or maturation process.

The Potassium Transporter Trk and External Potassium Modulate Salmonella enterica Protein Secretion and Virulence

ABSTRACT Potassium (K+) is the most abundant intracellular cation and is essential for many physiological functions of all living organisms; however, its role in the pathogenesis of human pathogens is not well understood. In this study, we characterized the functions of the bacterial Trk K+ transport system and external K+ in the pathogenesis of Salmonella enterica, a major food-borne bacterial pathogen. Here we report that Trk is important for Salmonella to invade and grow inside epithelial cells. It is also necessary for the full virulence of Salmonella in an animal infection model. Analysis of proteins of Salmonella indicated that Trk is involved in the expression and secretion of effector proteins of the type III secretion system (TTSS) encoded by Salmonella pathogenicity island 1 (SPI1) that were previously shown to be necessary for Salmonella invasion. In addition to the role of the Trk transporter in the pathogenesis of Salmonella, we discovered that external K+ modulates the pathogenic properties of Salmonella by increasing the expression and secretion of effector proteins of the SPI1-encoded TTSS and by enhancing epithelial cell invasion. Our studies demonstrated that K+ is actively involved in the pathogenesis of Salmonella and indicated that Salmonella may take advantage of the high K+ content inside host cells and in the intestinal fluid during diarrhea to become more virulent.

Molecular Characterization of Highly Pathogenic H5N1 Avian Influenza A Viruses Isolated from Raccoon Dogs in China

Background The highly pathogenic avian influenza H5N1 virus can infect a variety of animals and continually poses a threat to animal and human health. While many genotypes of H5N1 virus can be found in chicken, few are associated with the infection of mammals. Characterization of the genotypes of viral strains in animal populations is important to understand the distribution of different viral strains in various hosts. This also facilitates the surveillance and detection of possible emergence of highly pathogenic strains of specific genotypes from unknown hosts or hosts that have not been previously reported to carry these genotypes. Methodology/Principal Findings Two H5N1 isolates were obtained from lung samples of two raccoon dogs that had died from respiratory disease in China. Pathogenicity experiments showed that the isolates were highly pathogenic to chicken. To characterize the genotypes of these viruses, their genomic sequences were determined and analyzed. The genetic contents of these isolates are virtually identical and they may come from the same progenitor virus. Phylogenetic analysis indicated that the isolates were genetically closely related to genotype V H5N1 virus, which was first isolated in China in 2003, and were distinct from the dominant virus genotypes (e.g. genotype Z) of recent years. The isolates also contain a multibasic amino acid motif at their HA cleavage sites and have an E residue at position 627 of the PB2 protein similar to the previously-identified avian viruses. Conclusions/Significance This is the first report that genotype V H5N1 virus is found to be associated with a mammalian host. Our results strongly suggest that genotype V H5N1 virus has the ability to cross species barriers to infect mammalian animals. These findings further highlight the risk that avian influenza H5N1 virus poses to mammals and humans, which may be infected by specific genotypes that are not known to infect these hosts.

Survival characteristics of Salmonella enterica serovar Enteritidis in chicken egg albumen

Salmonella enterica serovar Enteritidis (SE) is a major foodborne pathogen primarily causing human infection through contaminated chicken eggs. To understand how SE survives in chicken egg albumen, we systematically and quantitatively analysed the survival properties of SE in egg albumen and identified factors affecting its survival. Survival assays of SE in egg indicate that egg albumen restricted the growth of SE. A major factor that controlled SEs growth in egg albumen was iron restriction, since egg albumen supplemented with iron allowed SE to grow, and iron acquisition mutants of SE showed decreased survival in egg albumen. In addition, low pH of albumen, high concentrations of bacteria and low incubation temperatures of bacteria with albumen facilitates the survival of SE. Our results suggest that egg albumen uses multiple mechanisms to control SE including iron limitation, surface interaction and possible enzymatic activities.