The emerging roles of long non-coding RNA in host immune response and intracellular bacterial infections.

The long non-coding RNAs (lncRNAs) are evolutionarily conserved classes of non-coding regulatory transcripts of > 200 nucleotides in length. They modulate several transcriptional and post-transcriptional events in the organism. Depending on their cellular localization and interactions, they regulate chromatin function and assembly; and alter the stability and translation of cytoplasmic mRNAs. Although their proposed range of functionality remains controversial, there is increasing research evidence that lncRNAs play a regulatory role in the activation, differentiation and development of immune signaling cascades; microbiome development; and in diseases such as neuronal and cardiovascular disorders; cancer; and pathogenic infections. This review discusses the functional roles of different lncRNAs in regulation of host immune responses, signaling pathways during host-microbe interaction and infection caused by obligate intracellular bacterial pathogens. The study of lncRNAs is assuming significance as it could be exploited for development of alternative therapeutic strategies for the treatment of severe and chronic pathogenic infections caused by Mycobacterium, Chlamydia and Rickettsia infections, as well as commensal colonization. Finally, this review summarizes the translational potential of lncRNA research in development of diagnostic and prognostic tools for human diseases.

Glucose Starvation, Magnesium Ion Starvation, and Bile Stress Assays.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a leading causative pathogen for food-borne gastroenteritis. During its course of infection, it confronts myriads of physiological barriers inside the host, such as nutrient deprivation, low micronutrient availability, and toxicity from bile salts, to promote bacterial survival and infection inside the host. The ability of the pathogen to overcome these stressful conditions determines the degree of virulence in the host. Therefore, assessment of the survival of a pathogen during different stress conditions, like glucose starvation, magnesium starvation, and bile stress, are important parameters to assess the virulence of the pathogen. Here, we describe protocols for estimating the survival of the pathogen during the above-mentioned stress conditions. We culture S. Enteritidis in an appropriate growth medium to a required O.D.600 and treat it with glucose starvation (M9 minimal culture medium containing 0.03% glucose), magnesium starvation (M9 minimal culture medium containing 20 µM MgSO4), and bile stress (bacterial cells treated with 15% bile salts in Luria Bertani (LB) culture medium) conditions. The number of surviving bacteria is obtained after the treatment by calculating the colony-forming units (CFU) of the surviving pathogen obtained on LB agar plates at relevant time intervals. The experiments are performed in biological replicates, and statistical analysis is performed to validate the experimental findings. The methodology of these stress response assays is simple and can be adapted to study the pathogenesis and stress response in other relevant and culturable enteric pathogens.

Role of OB-Fold Protein YdeI in Stress Response and Virulence of Salmonella enterica Serovar Enteritidis.

An essential feature of the pathogenesis of the Salmonella enterica serovar Enteritidis wild type (WT) is its ability to survive under diverse microenvironmental stress conditions, such as encountering antimicrobial peptides (AMPs) or glucose and micronutrient starvation. These stress factors trigger virulence genes carried on Salmonella pathogenicity islands (SPIs) and determine the efficiency of enteric infection. Although the oligosaccharide/oligonucleotide binding-fold (OB-fold) family of proteins has been identified as an important stress response and virulence determinant, functional information on members of this family is currently limited. In this study, we decipher the role of YdeI, which belongs to OB-fold family of proteins, in stress response and virulence of S Enteritidis. When ydeI was deleted, the ΔydeI mutant showed reduced survival during exposure to AMPs or glucose and Mg2+ starvation stress compared to the WT. Green fluorescent protein (GFP) reporter and quantitative real-time PCR (qRT-PCR) assays showed ydeI was transcriptionally regulated by PhoP, which is a major regulator of stress and virulence. Furthermore, the ΔydeI mutant displayed ∼89% reduced invasion into HCT116 cells, ∼15-fold-reduced intramacrophage survival, and downregulation of several SPI-1 and SPI-2 genes encoding the type 3 secretion system apparatus and effector proteins. The mutant showed attenuated virulence compared to the WT, confirmed by its reduced bacterial counts in feces, mesenteric lymph node (mLN), spleen, and liver of C57BL/6 mice. qRT-PCR analyses of the ΔydeI mutant displayed differential expression of 45 PhoP-regulated genes, which were majorly involved in metabolism, transport, membrane remodeling, and drug resistance under different stress conditions. YdeI is, therefore, an important protein that modulates S Enteritidis virulence and adaptation to stress during infection.IMPORTANCES Enteritidis during its life cycle encounters diverse stress factors inside the host. These intracellular conditions allow activation of specialized secretion systems to cause infection. We report a conserved membrane protein, YdeI, and elucidate its role in protection against various intracellular stress conditions. A key aspect of the study of a pathogen's stress response mechanism is its clinical relevance during host-pathogen interaction. Bacterial adaptation to stress plays a vital role in evolution of a pathogen's resistance to therapeutic agents. Therefore, investigation of the role of YdeI is vital for understanding the molecular basis of regulation of Salmonella pathogenesis. In conclusion, our findings may contribute to finding potential targets to develop new intervention strategies for treatment and prevention of enteric diseases.

RpoS-regulated SEN1538 gene promotes resistance to stress and influences Salmonella enterica serovar enteritidis virulence.

Salmonella enterica serovar Enteritidis (S. Enteritidis; wild type (WT)) is a major cause of foodborne illness globally. The ability of this pathogen to survive stress inside and outside the host, such as encountering antimicrobial peptides and heat stress, determines the efficiency of enteric infection. These stressors concertedly trigger virulence factors encoded on Salmonella pathogenicity islands (SPIs). Although RpoS is a well-known central transcriptional stress and virulence regulator, functional information regarding the genes of the regulon is currently limited. Here, we identified SEN1538 as a conserved RpoS-regulated gene belonging to the KGG protein superfamily. We further assessed its role in pathogenic stress responses and virulence. When SEN1538 was deleted (Δ1538), the pathogen showed reduced survival during antimicrobial peptide introduction and heat stress at 55°C compared to WT. The mutant displayed 70% reduced invasion in the HCT116 colon epithelial cell line, 5-fold attenuated phagocytic survival in RAW264.7 cells, and downregulation of several SPI-1 and SPI-2 genes encoding the three secretion system apparatus and effector proteins. Δ1538 also showed decreased virulence compared to WT, demonstrated by its reduced bacterial counts in the feces, mLN, spleen, and cecum of C57BL/6 mice. Comparative transcriptomic analysis of Δ1538 against WT revealed 111 differentially regulated genes, 103 of which were downregulated (fold change ≤ -1.5, P < 0.05). The majority of these genes were in clusters for metabolism, transporters, and pathogenesis, driving pathogenic stress responses and virulence. SEN1538 is, therefore, an important virulence determinant contributing to the resilience of S. Enteritidis to stress factors during infection.

A ROD9 island encoded gene in Salmonella Enteritidis plays an important role in acid tolerance response and helps in systemic infection in mice.

Salmonella, like other pathogenic bacteria has undergone multiple genomic alterations to adapt itself into specific host environments executing varied degrees of virulence through evolution. Such variations in genome content have been assumed to lead the closely related non-typhoidal serovars, S. Enteritidis, and S. Typhimurium to exhibit Type Three Secretion System -2 (T3SS-2) based diverse colonization and inflammation kinetics. Mutually exclusive genes present in either of the serovars are recently being studied and in our currentwork, we focused on a particular island ROD9, present in S. Enteritidis but not in S. Typhimurium. Earlier reports have identified a few genes from this island to be responsible for virulence in vitro as well as in vivo. In this study, we have identified another gene, SEN1008 from the same island encoding a hypothetical protein to be a potential virulence determinant showing systemic attenuation upon mutation in C57BL/6 mice infection model. The isogenic mutant strain displayed reduced adhesion to epithelial cells in vitro as well as was highly immotile. It was also deficient in intracellular replication in vitro, with a highly suppressed SPI-2and failed to cause acute colitis at 72-h p.i.in vivo. Moreover, on acid exposure, SEN1008 showed 17 folds and 2 fold up-regulations during adaptation and challenge phases,respectively and ΔSEN1008 failed to survive during ATR assay, indicating its role under acid stress. Together, our findings suggested ΔSEN1008 to be significantly attenuated and we propose this gene to be a potent factor responsible for S. Enteritidis pathogenesis.

"Omics" of Food-Borne Gastroenteritis: Global Proteomic and Mutagenic Analysis of Salmonella enterica Serovar Enteritidis.

Salmonella Enteritidis causes food-borne gastroenteritis by the two type three secretion systems (TTSS). TTSS-1 mediates invasion through intestinal lining, and TTSS-2 facilitates phagocytic survival. The pathogens' ability to infect effectively under TTSS-1-deficient background in host's phagocytes is poorly understood. Therefore, pathobiological understanding of TTSS-1-defective nontyphoidal Salmonellosis is highly important. We performed a comparative global proteomic analysis of the isogenic TTSS-1 mutant of Salmonella Enteritidis (M1511) and its wild-type isolate P125109. Our results showed 43 proteins were differentially expressed. Functional annotation further revealed that differentially expressed proteins belong to pathogenesis, tRNA and ncRNA metabolic processes. Three proteins, tryptophan subunit alpha chain, citrate lyase subunit alpha, and hypothetical protein 3202, were selected for in vitro analysis based on their functional annotations. Deletion mutants generated for the above proteins in the M1511 strain showed reduced intracellular survival inside macrophages in vitro. In sum, this study provides mass spectrometry-based evidence for seven hypothetical proteins, which will be subject of future investigations. Our study identifies proteins influencing virulence of Salmonella in the host. The study complements and further strengthens previously published research on proteins involved in enteropathogenesis of Salmonella and extends their role in noninvasive Salmonellosis.

Proteogenomics of Candida tropicalis--An Opportunistic Pathogen with Importance for Global Health.

The frequency of Candida infections is currently rising, and thus adversely impacting global health. The situation is exacerbated by azole resistance developed by fungal pathogens. Candida tropicalis is an opportunistic pathogen that causes candidiasis, for example, in immune-compromised individuals, cancer patients, and those who undergo organ transplantation. It is a member of the non-albicans group of Candida that are known to be azole-resistant, and is frequently seen in individuals being treated for cancers, HIV-infection, and those who underwent bone marrow transplantation. Although the genome of C. tropicalis was sequenced in 2009, the genome annotation has not been supported by experimental validation. In the present study, we have carried out proteomics profiling of C. tropicalis using high-resolution Fourier transform mass spectrometry. We identified 2743 proteins, thus mapping nearly 44% of the computationally predicted protein-coding genes with peptide level evidence. In addition to identifying 2591 proteins in the cell lysate of this yeast, we also analyzed the proteome of the conditioned media of C. tropicalis culture and identified several unique secreted proteins among a total of 780 proteins. By subjecting the mass spectrometry data derived from cell lysate and conditioned media to proteogenomic analysis, we identified 86 novel genes, 12 novel exons, and corrected 49 computationally-predicted gene models. To our knowledge, this is the first high-throughput proteomics study of C. tropicalis validating predicted protein coding genes and refining the current genome annotation. The findings may prove useful in future global health efforts to fight against Candida infections.

The O-antigen negative ∆wbaV mutant of Salmonella enterica serovar Enteritidis shows adaptive resistance to antimicrobial peptides and elicits colitis in streptomycin pretreated mouse model.

BACKGROUND: Salmonella enterica serovar Enteritidis, the most common cause of human gastroenteritis, employs several virulence factors including lipopolysaccharide (LPS) for infection and establishment of disease inside the host. The LPS of S. enterica serovar Enteritidis consists of lipid A, core oligosaccharide and O-antigen (OAg). The OAg consists of repeating units containing different sugars. The sugars of OAg are synthesized and assembled by a set of enzymes encoded by genes organized into clusters. Present study focuses on the effect of deletion of genes involved in biosynthesis of OAg repeating units on resistance to antimicrobial peptides and virulence in mice. METHODS: In the present study, the OAg biosynthesis was impaired by deleting tyv, prt and wbaV genes involved in tyvelose biosynthesis and its transfer to OAg. The virulence phenotype of resulting mutants was evaluated by assessing resistance to antimicrobial peptides, serum complement, adhesion, invasion and in vivo colonization. RESULTS: Deletion of the above three genes resulted in the production of OAg-negative LPS. All the OAg-negative mutants showed phenotype reported for rough strains. Interestingly, ΔwbaV mutant showed increased resistance against antimicrobial peptides and normal human serum. In addition, the ΔwbaV mutant also showed increased adhesion and invasion as compared to the other two O-Ag negative mutants Δtyv and Δprt. In vivo experiments also confirmed the increased virulent phenotype of ΔwbaV mutant as compared to Δprt mutant. CONCLUSION: OAg-negative mutants are known to be avirulent; however, this study demonstrates that certain OAg negative mutants e.g. ∆wbaV may also show resistance to antimicrobial peptides and cause colitis in Streptomyces pretreated mouse model.