Recoding anaerobic regulator fnr of Salmonella Typhimurium attenuates it's pathogenicity.

AIMS: How recoding of fnr, an anaerobic regulatory gene, affects pathogenicity related parameters of Salmonella Typhimurium (STM). METHODS AND RESULTS: The fnr gene was recoded by substituting all of it's codons with synonymous rare codons of STM. Recoding fnr gene severely reduced the ability of the recoded mutant to compete with wild strain under nutrient depletion condition. Mutants were also less motile than the wild strain and their biofilm forming ability was significantly decreased as compared to wild strain. The recoded strain showed significant reduced survival within murine macrophages (RAW264.7) and monocyte derived macrophage of poultry origin. The colonisation ability of recoded mutant in liver and spleen of mice on day 5 of post infection was significantly reduced. The recoded strain exhibited significant reduction in faecal shedding on day 1 and 5 after infection. CONCLUSIONS: Our study showed that recoding the anaerobic regulator fnr of STM significantly compromised its growth, decreased motility, biofilm forming ability and survival within macrophages. Further, the recoded fnr strain showed reduced colonisation ability and faecal shedding in mice. Thus, these findings highlight that recoding the global anaerobic regulator fnr of Salmonella Typhimurium attenuates its pathogenicity.

Comparative proteomic analysis of Salmonella Typhimurium wild type and its isogenic fnr null mutant during anaerobiosis reveals new insight into bacterial metabolism and virulence.

AIM: The aim of this study was to understand the role of anaerobic regulator FNR (Fumarate Nitrate Reduction) in Salmonella Typhimurium through proteomic approach. METHODS AND RESULTS: We did label free quantitative proteomic analysis of Salmonella Typhimurium PM45 wild type and the fnr null mutant cultured under anaerobic conditions. The data revealed 153 significantly differentially expressed proteins (DEPs) in the mutant out of 1798 total proteins identified. Out of 153 DEPs, 94 proteins were up-regulated (repressed by FNR) and 59 proteins were down-regulated (activated by FNR) in the mutant. The network analysis indicated up-regulation of TCA cycle, electron transport chain and ethanolamine metabolism and down regulation of pyruvate metabolism and glycerol and glycerophospholipid metabolism. CONCLUSIONS: Our study showed that FNR represses ethanolamine utilization. The different metabolic pathways such as pyruvate metabolism, glycerol metabolism and glycerophospholipid metabolism were activated by FNR. Further, FNR positively regulated the DNA binding protein Fis, one of the global regulators of virulence in Salmonella Typhimurium. Thus, our finding highlights the pivotal role of FNR in regulating bacterial metabolism and virulence during anaerobiosis for systemic infection of the host.

Systematic evaluation of species-independent serum pre-fractionation strategies revealed cost-effective methods to reduce proteome complexity.

In this study, the efficiency of one commercial (ProteoMiner™ -PM) and five simple and cost-effective laboratory chemicals (Acetone, TCA/acetone, DTT, ACN and DTT-ACN) based serum protein pre-fractionation strategies was compared in pig model by label-free quantitation based mass spectrometric approach to find out the most suitable strategy for reducing the complexity of serum proteome for subsequent proteomic studies. The highest serum protein depletion percentage and highest depletion of albumin, the most abundant serum protein, was observed in DTT-ACN method. The maximum number of serum proteins was identified in ACN followed by DTT-ACN method and importantly, detection of more number of low-abundant proteins (LAPs) could also be achieved by these two methods. Although PM method resulted into lowest dynamic range of protein abundance, quite a less number of proteins were identified by this method. Overall, sequential depletion using DTT-ACN and ACN methods provided advantage of simultaneous detection of more number of proteins along with LAPs with a reasonably high dynamic range of protein abundances over other methods and thus emerged as cheaper and effective alternatives to the commercial methods. Further, these methods are species-independent and hence can be applied in human and in any livestock species to simplify the serum proteome.

Comparative serum proteome analysis reveals potential early pregnancy-specific protein biomarkers in pigs.

In this study, the comparative serum proteome profile of Day 5, 12 and 16 of gestation, representing three early embryonic events, namely formation, elongation and implantation of blastocysts, and non-pregnant control were explored by a label-free quantitation-based mass spectrometric approach to identify early pregnancy biomarkers in pigs. A total of 131 proteins were identified with respect to different groups, out of which 105 were found to be differentially expressed proteins (DEPs). Among the DEPs, 54 and 66 proteins were found to be up and downregulated respectively in early pregnancy groups (fold change >2) and the maximum number of upregulated proteins was observed in the Day 12 pregnancy stage. Functional classification and pathway analysis of the DEPs revealed involvement of most of the proteins in complement and coagulation cascades, metabolic processes and immune and inflammatory responses. Proteins such as glutathione peroxidise (GPX), pregnancy zone protein (PZP), thrombospondin-1 (THBS1), α-1-antitrypsin (AAT) and mannose-binding lectin C (MBLC) were differentially expressed during early pregnancy and actively involved in different pregnancy-related activities. To the best of our knowledge, this is the first report on comparative serum protein profiling of different early pregnancy stages in pigs and our results provide a set of proteins that can be used as potential biomarkers for early pregnancy diagnosis in pigs.

Isolation and molecular characterization of GP5 glycoprotein gene of Betaarterivirus suid 2 from Mizoram, India.

Porcine reproductive and respiratory syndrome (PRRS) is a serious swine disease causing great economic impact worldwide. The emergence of highly pathogenic strains in Asian countries is associated with large scale mortality in all age groups of pigs besides the classical presentation of severe respiratory distress, pneumonia, and a series of reproductive disorders in sows, like late-term abortion, premature farrowing, and an increased number of stillborn piglets. The present study was designed with the aim of isolation and characterization of the Betaarterivirus suid 2 from outbreaks in Mizoram in primary porcine alveolar macrophage and subsequently characterized the GP5 gene sequence of the isolate in terms of phylogenetic analysis and deduce amino acid sequence comparison. Virus propagation was performed in the porcine alveolar macrophage (PAM) primary cell culture and confirmed by immunoperoxidase test, FAT, and nested RT-PCR. The full-length GP5 gene (603nt) was amplified from the isolate and subsequently cloned and sequenced (MN928985). Phylogenetic analysis and sequence comparison of the present isolate was found to have similarity 98.7-98.8% with Myanmar HP-PRRS strains, 98-98.5% with Vietnam strains, 98.2-98.3% with China strains, indicating a close lineage with highly pathogenic PRRS strains. In deduced amino acid sequence analysis, one mutation was found in the primary neutralizing epitope (PNE) at position 39L → I39 and one more mutation was also found in the decoy epitope (DCE) at position 30 N → D30. The amino acid at this position is an N-linked glycosylation site, and mutation of the N-linked glycosylation is an immune escaped strategy adopted by this virus causing a persistent infection in the natural host.

Dynamics of cytokine gene expression in peripheral blood mononuclear cells of indigenous and exotic breeds of pigs in India.

To incorporate immune competence traits in swine breeding programs, association between immune responsiveness and susceptibility to specific infectious diseases must be established. In order to understand the differences in immune competence between indigenous (Zovawk) and exotic (Large White Yorkshire: LWY) pigs reared in India, we carried out a time course expression analysis of immune-regulating key cytokine genes (interleukin (IL)-2, interferon (IFN)-γ, IL-4 and IL-10) in the phytohemagglutinin-P stimulated peripheral blood mononuclear cells (PBMCs). The IL-2 transcript levels in PBMCs increased several thousand-fold when compared to unstimulated cells in both the breeds, albeit the response in that of Zovawk was remarkably higher. Higher and earlier IFN-γ and IL-4 expression levels in Zovawk pigs suggest that both TH 1 and TH 2 immune responsiveness of this indigenous breed affords better preparedness for danger signals. Moreover, the low expression levels of IL-10 depict a regulated adaptive immune responsiveness. Remarkable difference between the two breeds of the pigs is evident showing a clear advantage of the Zovawk over LWY in terms of a shorter lag period of adaptive immune response. These findings provide a lead for understanding the genetic differences with respect to immune competence levels of indigenous pigs compared to exotic counterparts.