Syed Imteyaz Alam

Comparative Proteomic Analysis of Extracellular Proteins of Clostridium perfringens Type A and Type C Strains

ABSTRACT Clostridium perfringens is a medically important clostridial pathogen and an etiological agent causing several diseases in humans and animals. C. perfringens and its toxins have been listed as potential biological and toxin warfare (BTW) agents; thus, efforts to develop strategies for detection and protection are warranted. Forty-eight extracellular proteins of C. perfringens type A and type C strains have been identified here using a 2-dimensional gel electrophoresis-mass spectrometry (2-DE-MS) technique. The SagA protein, the DnaK-type molecular chaperone hsp70, endo-beta-N-acetylglucosaminidase, and hypothetical protein CPF_0656 were among the most abundant proteins secreted by C. perfringens ATCC 13124. The antigenic component of the exoproteome of this strain has also been identified. Most of the extracellular proteins were predicted to be involved in carbohydrate transport and metabolism (16%) or cell envelope biogenesis or to be outer surface protein constituents (13%). More than 50% of the proteins were predictably secreted by either classical or nonclassical pathways. LipoP and TMHMM indicated that nine proteins were extracytoplasmic but cell associated. Immunization with recombinant ornithine carbamoyltransferase (cOTC) clearly resulted in protection against a direct challenge with C. perfringens organisms. A significant rise in IgG titers in response to recombinant cOTC was observed in mice, and IgG2a titers predominated over IgG1 titers (IgG2a/IgG1 ratio, 2). The proliferation of spleen lymphocytes in cOTC-immunized animals suggested a cellular immune response. There were significant increases in the levels of gamma interferon (IFN-γ) and interleukin 2 (IL-2), suggesting a Th1 type immune response.

Effect of Iron, Nickel and Cobalt on Bacterial Activity and Dynamics During Anaerobic Oxidation of Organic Matter

The present study reports on the effect of Fe2+, Ni2+ and CO2+ on anaerobic bacterial activity and dynamics during anaerobic mineralization of organic matter. Fe2+ stimulated biogas yield and methane content at 37 °C, but inhibited same marginally at low temperatures (10 and 20 °C). Ni2+ and CO2+ inhibited methane production; the toxicity increased with decreasing temperature. Fe2+ stimulated sulfate reducing bacterial (SRB) and methane producing bacterial (MPB) counts while Ni2+ and CO2+ inhibited the same. All the metals had no effect on obligate proton reducing bacterial (OPRB) counts. Fe2+ stimulated, Ni2+ severely and CO2+ appreciably inhibited H2 uptake and acetate/propionate utilization irrespective of presence/absence of sulfate. Thus, Ni2+ and CO2+ inhibited the overall anaerobic degradative process by inhibiting SRB and MPB activity.

Proteolytic anaerobic bacteria from lake sediments of Antarctica

Amongst twenty five proteolytic bacteria isolated from lake sediment samples of Antarctica, six isolates were selected based on SDS PAGE protein profile and zone of hydrolysis on casein agar at 10 degrees C. Most of the cultures were rod shaped and motile with two showing terminal bulging spores. Isolates grew between 5 degrees C to 37 degrees C and protease was induced in the late log, stationary or death phase. Isolate SPA-3 grew maximally at 10 degrees C and SPA-6 at 37 degrees C while others preferred 20 degrees C-30 degrees C for growth. The growth and protease production on casein, skimmed milk, bovine serum albumin and gelatin varied with the isolates. Acetate was the dominant volatile fatty acid (24-66% of total VFA) produced during hydrolysis of protein substrate.

Differential proteomic analysis of Clostridium perfringens ATCC13124; identification of dominant, surface and structure associated proteins

BackgroundClostridium perfringens is a medically important clostridial pathogen causing diseases in man and animals. To invade, multiply and colonize tissues of the host, a pathogen must be able to evade host immune system, and obtain nutrients essential for growth. The factors involved in these complex processes are largely unknown and of crucial importance to understanding microbial pathogenesis. Many of the virulence determinants and putative vaccine candidates for bacterial pathogens are known to be surface localized.ResultsUsing 2-DE mass spectrometry strategy, we identified major surface (22) and cell envelope (10) proteins from Clostridium perfringens ATCC13124 and those differentially expressed (11) in cells grown on cooked meat medium (CMM) in comparison with cells grown in reference state (tryptose-yeast extract-glucose medium). Riboflavin biosynthesis protein, ornithine carbamoyltransferase, cystathionine beta-lyase, and threonine dehydratase were the predominant proteins that exhibited 2.19 to 8.5 fold increase in the expression level in cells growing on CMM.ConclusionOrnithine carbamoyltransferase and cystathionine beta-lyase were over-expressed in cells grown on cooked meat medium and also identified in the surface protein fraction and the former was immunogenic; making them potential vaccine candidates. Based upon bioinformatic analysis; choloylglycine hydrolase family protein, cell wall-associated serine proteinase, and rhomboid family protein were predicted as surface protein markers for specific detection of C. perfringens from the environment and food. Most of the proteins over-expressed in CMM were shown to have putative function in metabolism, of which seven were involved in amino acid transport and metabolism or lipid metabolism.

Psychrophilic Planococcus maitriensis sp.nov. from Antarctica.

An orange pigmented bacterium, S1, was isolated from a cyanobacterial mat sample collected in the vicinity of Schirmacher Oasis, Maitri, the Indian station, in Antarctica. The bacterium is Gram-positive and possesses all the characteristics of the genus Planococcus. It is non-sporulating, motile and has A4alpha type peptidoglycan, MK-7 and MK-8 as the major menaquinones and anteiso-C(15:0) as the major fatty acid. Based on the phylogenetic characteristics, the bacterium S1 is identified as a close relative of Planococcus citreus with which it shares 98.12% similarity at the 16S rRNA gene level but exhibits a low similarity of 52% at the whole genome level. Apart from the above major differences, S1 also exhibits phenotypic differences with Planococcus citreus and other members of the genus Planococcus. Based on these differences, the bacterium S1 is identified as a new species of the genus Planococcus for which the name Planococcus maitriensis is proposed. The type strain of Planococcus maitriensis is S1(T) (= MTCC 4827; DSM 15305).

Diversity and antibiotic susceptibility pattern of cultivable anaerobic bacteria from soil and sewage samples of India

Soil and sewage act as a reservoir of animal pathogens and their dissemination to animals profoundly affects the safety of our food supply. Moreover, acquisition and further spread of antibiotic resistance determinants among pathogenic bacterial populations is the most relevant problem for the treatment of infectious diseases. Bacterial strains from soil and sewage are a potential reservoir for antimicrobial resistance genes. Accurate species determination for anaerobes from environmental samples has become increasingly important with the re-emergence of anaerobic bacteremia and prevalence of multiple-drug-resistant microorganisms. Soil samples were collected from various locations of planar India and the diversity of anaerobic bacteria was determined by 16S rRNA gene sequencing. Viable counts of anaerobic bacteria on anaerobic agar and SPS agar ranged from 1.0 × 10(2)cfu/g to 8.8 × 10(7)cfu/g and nil to 3.9 × 10(6)cfu/g, respectively. Among clostrdia, Clostridium bifermentans (35.9%) was the most dominant species followed by Clostridium perfringens (25.8%). Sequencing and phylogenetic analysis of C. perfringens beta2 toxin gene (cpb2) fragment indicated specific phylogenetic affiliation with cluster Ia for 5 out of 6 strains. Antibiotic susceptibility for 30 antibiotics was tested for 74 isolates, revealing resistance for as high as 16-25 antibiotics for 35% of the strains tested. Understanding the diversity of the anaerobic bacteria from soil and sewage with respect to animal health and spread of zoonotic pathogen infections is crucial for improvements in animal and human health.

Biogas production from night soil. Effects of loading and temperature

Anaerobic digestion of night soil was carried out in 25-litre digeters at 20°C. Digesters with 20–25 days HRT produced 23–28 litre biogas day−1 with 60–63% methane content. The volatile fatty acids and pH were maintained below 800 ppm and around 7·3, respectively. Batch studies at 5–30°C showed increased biogas production with temperature, and with volatile solids up to 6·2%. Volatile fatty acids, in general, increased directly with volatile solids and inversely with temperature. Microbial counts and enzyme activities in the stabilized digesters are also reported.

Production of biogas from night soil at psychrophilic temperature.

Abstract Anaerobic digestion of night soil was carried out in a 25 l digester at 10°C using an adapted inoculum. Biogas production was studied at 20, 25, 30, 35 and 40 days hydraulic retention time. Digesters operated at 25 day hydraulic retention time produced 69·72 l biogas/kg VS/day with a methane content of 73·0%. Microbial counts and enzyme activities are also reported.

Host response to intravenous injection of epsilon toxin in mouse model: A proteomic view

Epsilon toxin (ETX) is an extremely potent pore‐forming toxin and a category B biological agent. ETX is a major virulence determinant of Clostridium perfringens toxinotypes B and D, and is implicated in pathogenesis of rapidly fatal economically important pulpy kidney disease in lambs caused by toxinotype D. Despite being a toxin, ETX can be utilized as a tool to target glutamatergic neurons and for drug delivery into the CNS. 2DE‐MS approach was employed to elucidate the host response to ETX following intravenous injection in mouse model. In total, 136 proteins were identified either differentially expressed in brain (18) and kidney (33); showing specific interaction with ETX from lysates of brain (4), kidney (21), or from plasma (42); and urine markers (18) of intoxication. Differentially expressed proteins in kidney included those involved in calcium homeostasis and cytoskeletal organization. Proteins involved in ER and oxidative stress and energy metabolism also showed differential levels in the target tissue after ETX treatment. The known functions of the proteins differentially expressed and those interacting with ETX indicate involvement of interlinked pathways. This study provides first proteomic account of host response to ETX exposure providing clues to mechanism of toxicity and potential therapeutic targets.

Effect of propionate toxicity on methanogenesis of night soil at phychrophilic temperature.

The effect of propionate concentrations on biodegradation of human waste (night soil) was studied at 10 degrees C. Propionate was toxic for the biomethanation at all the pH tested (6.0, 7.0 and 8.0). The maximum reduction in biogas production in presence of 200 mM propionate was observed at pH 7.0 followed by 8.0. The methane content in biogas also followed a similar trend and at pH 7.0 an 11.5% decrease was observed. Propionate caused the reduction of methanogenic count by an approximately 2log value. Total volatile fatty acids increased with the increase in propionate concentration and particularly accumulation of propionate was observed. The results were also compared with the 30 degrees C fermentation.