Klebsiella pneumoniae C o-infection Leads to Fatal Pneumonia in SARS-CoV-2-infected Mice.

SARS-CoV-2 patients have been reported to have high rates of secondary Klebsiella pneumoniae infections. Klebsiella pneumoniae is a commensal that is typically found in the respiratory and gastrointestinal tracts. However, it can cause severe disease when a person's immune system is compromised. Despite a high number of K. pneumoniae cases reported in SARS-CoV-2 patients, a co-infection animal model evaluating the pathogenesis is not available. We describe a mouse model to study disease pathogenesis of SARS-CoV-2 and K. pneumoniae co-infection. BALB/cJ mice were inoculated with mouse-adapted SARS-CoV-2 followed by a challenge with K. pneumoniae . Mice were monitored for body weight change, clinical signs, and survival during infection. The bacterial load, viral titers, immune cell accumulation and phenotype, and histopathology were evaluated in the lungs. The co-infected mice showed severe clinical disease and a higher mortality rate within 48 h of K. pneumoniae infection. The co-infected mice had significantly elevated bacterial load in the lungs, however, viral loads were similar between co-infected and single-infected mice. Histopathology of co-infected mice showed severe bronchointerstitial pneumonia with copious intralesional bacteria. Flow cytometry analysis showed significantly higher numbers of neutrophils and macrophages in the lungs. Collectively, our results demonstrated that co-infection of SARS-CoV-2 with K. pneumoniae causes severe disease with increased mortality in mice.

Characterization of three Francisella tularensis genomes from Oklahoma, USA.

Francisella tularensis , the causative agent for tularaemia, is a Tier 1 select agent, and a pan-species pathogen of global significance due to its zoonotic potential. Consistent genome characterization of the pathogen is essential to identify novel genes, virulence factors, antimicrobial resistance genes, for studying phylogenetics and other features of interest. This study was conducted to understand the genetic variations among genomes of F. tularensis isolated from two felines and one human source. Pan-genome analysis revealed that 97.7 % of genes were part of the core genome. All three F. tularensis isolates were assigned to sequence type A based on single nucleotide polymorphisms (SNPs) in sdhA. Most of the virulence genes were part of the core genome. An antibiotic resistance gene coding for class A beta-lactamase was detected in all three isolates. Phylogenetic analysis showed that these isolates clustered with other isolates reported from Central and South-Central USA. Assessment of large sets of the F. tularensis genome sequences is essential in understanding pathogen dynamics, geographical distribution and potential zoonotic implications.

Development and evaluation of Microbe Finder (MiFi)®: a novel in silico diagnostic platform for pathogen detection from metagenomic data.

Introduction. With expanding demand for diagnostics, newer methodologies are needed for faster, user-friendly and multiplexed pathogen detection. Metagenome-based diagnostics offer potential solutions to address these needs as sequencing technologies have become affordable. However, the diagnostic utility of sequencing technologies is currently limited since analysis of the large amounts of data generated, are either computationally expensive or carry lower sensitivity and specificity for pathogen detection.Hypothesis/Gap Statement. There is a need for novel, user friendly, and computationally inexpensive platforms for metagenome sequence analysis for diagnostic applications.Methods. In this study, we report the use of MiFi® (Microbe Finder), a computationally inexpensive algorithm with a user-friendly online interface, for accurate, rapid and multiplexed pathogen detection from metagenome sequence data. Detection is accomplished based on identification of signature genomic sequence segments of the target pathogen in metagenome sequence data. In this study we used bovine respiratory disease (BRD) complex as a model.Results and Conclusions. Using MiFi®, multiple target bacteria and a DNA virus were successfully detected in a multiplex format from metagenome sequences acquired from bovine lung tissue. Overall, 51 clinical samples were assessed and MiFi® showed 100 % analytical specificity and varying levels of analytical sensitivity (62.5 %-100 %) when compared with other traditional pathogen detection techniques, such as PCR. Consistent detection of bacteria was possible from lung samples artificially spiked with 109-104 c.f.u. of Mannheimia haemolytica.

SARS CoV-2 (Delta Variant) Infection Kinetics and Immunopathogenesis in Domestic Cats.

Continued emergence of SARS-CoV-2 variants highlights the critical need for adaptable and translational animal models for acute COVID-19. Limitations to current animal models for SARS CoV-2 (e.g., transgenic mice, non-human primates, ferrets) include subclinical to mild lower respiratory disease, divergence from clinical COVID-19 disease course, and/or the need for host genetic modifications to permit infection. We therefore established a feline model to study COVID-19 disease progression and utilized this model to evaluate infection kinetics and immunopathology of the rapidly circulating Delta variant (B.1.617.2) of SARS-CoV-2. In this study, specific-pathogen-free domestic cats (n = 24) were inoculated intranasally and/or intratracheally with SARS CoV-2 (B.1.617.2). Infected cats developed severe clinical respiratory disease and pulmonary lesions at 4- and 12-days post-infection (dpi), even at 1/10 the dose of previously studied wild-type SARS-CoV-2. Infectious virus was isolated from nasal secretions of delta-variant infected cats in high amounts at multiple timepoints, and viral antigen was co-localized in ACE2-expressing cells of the lungs (pneumocytes, vascular endothelium, peribronchial glandular epithelium) and strongly associated with severe pulmonary inflammation and vasculitis that were more pronounced than in wild-type SARS-CoV-2 infection. RNA sequencing of infected feline lung tissues identified upregulation of multiple gene pathways associated with cytokine receptor interactions, chemokine signaling, and viral protein-cytokine interactions during acute infection with SARS-CoV-2. Weighted correlation network analysis (WGCNA) of differentially expressed genes identified several distinct clusters of dysregulated hub genes that are significantly correlated with both clinical signs and lesions during acute infection. Collectively, the results of these studies help to delineate the role of domestic cats in disease transmission and response to variant emergence, establish a flexible translational model to develop strategies to prevent the spread of SARS-CoV-2, and identify potential targets for downstream therapeutic development.

Multi-laboratory evaluation of the Illumina iSeq platform for whole genome sequencing of Salmonella, Escherichia coli and Listeria.

There is a growing need for public health and veterinary laboratories to perform whole genome sequencing (WGS) for monitoring antimicrobial resistance (AMR) and protecting the safety of people and animals. With the availability of smaller and more affordable sequencing platforms coupled with well-defined bioinformatic protocols, the technological capability to incorporate this technique for real-time surveillance and genomic epidemiology has greatly expanded. There is a need, however, to ensure that data are of high quality. The goal of this study was to assess the utility of a small benchtop sequencing platform using a multi-laboratory verification approach. Thirteen laboratories were provided the same equipment, reagents, protocols and bacterial reference strains. The Illumina DNA Prep and Nextera XT library preparation kits were compared, and 2×150 bp iSeq i100 chemistry was used for sequencing. Analyses comparing the sequences produced from this study with closed genomes from the provided strains were performed using open-source programs. A detailed, step-by-step protocol is publicly available via protocols.io (https://www.protocols.io/view/iseq-bacterial-wgs-protocol-bij8kcrw). The throughput for this method is approximately 4-6 bacterial isolates per sequencing run (20-26 Mb total load). The Illumina DNA Prep library preparation kit produced high-quality assemblies and nearly complete AMR gene annotations. The Prep method produced more consistent coverage compared to XT, and when coverage benchmarks were met, nearly all AMR, virulence and subtyping gene targets were correctly identified. Because it reduces the technical and financial barriers to generating WGS data, the iSeq platform is a viable option for small laboratories interested in genomic surveillance of microbial pathogens.

Clinical and Histopathologic Features of a Feline SARS-CoV-2 Infection Model Are Analogous to Acute COVID-19 in Humans.

The emergence and ensuing dominance of COVID-19 on the world stage has emphasized the urgency of efficient animal models for the development of therapeutics for and assessment of immune responses to SARS-CoV-2 infection. Shortcomings of current animal models for SARS-CoV-2 include limited lower respiratory disease, divergence from clinical COVID-19 disease, and requirements for host genetic modifications to permit infection. In this study, n = 12 specific-pathogen-free domestic cats were infected intratracheally with SARS-CoV-2 to evaluate clinical disease, histopathologic lesions, and viral infection kinetics at 4 and 8 days post-inoculation; n = 6 sham-inoculated cats served as controls. Intratracheal inoculation of SARS-CoV-2 produced a significant degree of clinical disease (lethargy, fever, dyspnea, and dry cough) consistent with that observed in the early exudative phase of COVID-19. Pulmonary lesions such as diffuse alveolar damage, hyaline membrane formation, fibrin deposition, and proteinaceous exudates were also observed with SARS-CoV-2 infection, replicating lesions identified in people hospitalized with ARDS from COVID-19. A significant correlation was observed between the degree of clinical disease identified in infected cats and pulmonary lesions. Viral loads and ACE2 expression were also quantified in nasal turbinates, distal trachea, lungs, and other organs. Results of this study validate a feline model for SARS-CoV-2 infection that results in clinical disease and histopathologic lesions consistent with acute COVID-19 in humans, thus encouraging its use for future translational studies.

Airborne transmission of virus-laden aerosols inside a music classroom: Effects of portable purifiers and aerosol injection rates.

The ongoing COVID-19 pandemic has shifted attention to the airborne transmission of exhaled droplet nuclei within indoor environments. The spread of aerosols through singing and musical instruments in music performances has necessitated precautionary methods such as masks and portable purifiers. This study investigates the effects of placing portable air purifiers at different locations inside a classroom and the effects of different aerosol injection rates (e.g., with and without masks, different musical instruments, and different injection modes). Aerosol deposition, airborne concentration, and removal are analyzed in this study. It was found that using purifiers could help in achieving ventilation rates close to the prescribed values by the World Health Organization, while also achieving aerosol removal times within the Center of Disease Control and Prevention recommended guidelines. This could help in deciding break periods between classroom sessions, which was around 25 min through this study. Moreover, proper placement of purifiers could offer significant advantages in reducing airborne aerosol numbers (offering several orders of magnitude higher aerosol removal when compared to nearly zero removal when having no purifiers), and improper placement of the purifiers could worsen the situation. This study suggests the purifier to be placed close to the injector to yield a benefit and away from the people to be protected. The injection rate was found to have an almost linear correlation with the average airborne aerosol suspension rate and deposition rate, which could be used to predict the trends for scenarios with other injection rates.

Virome characterization in serum of healthy show pigs raised in Oklahoma demonstrated great diversity of ssDNA viruses.

In the United States, show pigs are raised to compete in agricultural events. These animals are usually raised in small herds with extensive human, domestic, and wild animal contact. Therefore, pathogen monitoring in this animal category is critical for improved disease surveillance and preparedness. This study characterized the virome of healthy show pigs using high-throughput sequencing using pooled serum samples from 2018 or 2019 (200 samples each pool). Results demonstrated the presence of DNA viral families (Parvoviridae, Circoviridae, and Herpesviridae) and RNA families (Arteriviridae, Flaviviridae, and Retroviridae). Twenty-three viral species were identified, including the first detection of porcine bufavirus in the US. Moreover, important swine pathogens identified included porcine reproductive and respiratory syndrome virus, atypical porcine pestivirus, and porcine circovirus (PCV). Additionally, complete coding genomes of 17 viruses from the Parvoviridae, Anelloviridae, and Circoviridae families were retrieved and included the first near full-length genomes of US Ungulate bocaparvovirus 3 species.

Pooling of Nasopharyngeal Swab Samples To Overcome a Global Shortage of Real-Time Reverse Transcription-PCR COVID-19 Test Kits.

The global outbreak and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have created an urgent need for large-scale testing of populations. There is a demand for high-throughput testing protocols that can be used for efficient and rapid testing of clinical specimens. We evaluated a pooled PCR protocol for testing nasopharyngeal (NP) swabs using known positive/negative and untested clinical samples that were assigned to pools of 5 or 10. In total, 630 samples were used in this study. Individual positive samples with cycle threshold (CT ) values as high as 33 could be consistently detected when pooled with 4 negative samples (pool of 5), and individual positive samples with CT values up to 31 could be consistently detected when pooled with 9 negative samples (pool of 10). Pooling of up to 5 samples can be employed in laboratories for the diagnosis of COVID-19 for efficient utilization of resources, rapid screening of a greater number of people, and faster reporting of test results.

Safety and efficacy of cold atmospheric plasma for the sterilization of a Pasteurella multocida-contaminated subcutaneously implanted foreign body in rabbits.

OBJECTIVE: To determine whether a stainless steel implant sterilized with a novel cold atmospheric plasma sterilization (CAPS) device adversely affects local tissues in rabbits and whether CAPS was as effective as steam sterilization with an autoclave to inactivate Pasteurella multocida. ANIMALS: 31 healthy New Zealand White rabbits. PROCEDURES: Steam-autoclaved stainless steel implants inoculated with P multocida underwent a second steam autoclave sterilization (AIA) or CAPS (AICAPS). One AIA implant and 3 AICAPS implants were randomly placed subcutaneously at 4 sites in 21 rabbits (84 implants). These rabbits were monitored daily for 5 days for evidence of systemic illness and local tissue reactions at the implantation sites and then euthanized. Samples were taken from each implant site for bacterial culture and histologic examination. RESULTS: Cultures of samples obtained from all sites were negative for bacterial growth. No significant difference was observed in mean skin thickness or erythema between AIA and AICAPS implant sites on any observed day. Also, individual histologic grades for the epidermis, dermis, subcutis, and muscle and total histologic grade were not significantly different between AIA and AICAPS implant sites. CONCLUSIONS AND CLINICAL RELEVANCE: Cold atmospheric plasma sterilization was noninferior to steam sterilization of P multocida-contaminated stainless steel implants in the rabbits in the present study. However, studies of the efficacy of CAPS for inactivation of other important bacteria are needed.

SARS-CoV-2 Genomes From Oklahoma, United States.

Genomic sequencing has played a major role in understanding the pathogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the current pandemic, it is essential that SARS-CoV-2 viruses are sequenced regularly to determine mutations and genomic modifications in different geographical locations. In this study, we sequenced SARS-CoV-2 from five clinical samples obtained in Oklahoma, United States during different time points of pandemic presence in the state. One sample from the initial days of the pandemic in the state and four during the peak in Oklahoma were sequenced. Previously reported mutations including D614G in S gene, P4715L in ORF1ab, S194L, R203K, and G204R in N gene were identified in the genomes sequenced in this study. Possible novel mutations were also detected in the S gene (G1167V), ORF1ab (A6269S and P3371S), ORF7b (T28I), and ORF8 (G96R). Phylogenetic analysis of the genomes showed similarity to other SARS-CoV-2 viruses reported from across the globe. Structural characterization indicates that the mutations in S gene possibly influences conformational flexibility and motion of the spike protein, and the mutations in N gene are associated with disordered linker region within the nucleocapsid protein.

Whole-Genome Sequence of Multidrug-Resistant Bibersteinia trehalosi Strain OADDL-BT1.

The genome of a multidrug-resistant strain of Bibersteinia trehalosi isolated from a calf with chronic pneumonia is presented. The draft genome sequences have been deposited at DDBJ/ENA/GenBank.

Investigation of the Roles of Plasma Species Generated by Surface Dielectric Barrier Discharge.

As an emerging sterilization technology, cold atmospheric plasma offers a dry, non-thermal, rapid process that is minimally damaging to a majority of substrates. However, the mechanisms by which plasma interacts with living cells are poorly understood and the plasma generation apparatuses are complex and resource-intensive. In this study, the roles of reactive oxygen species (ROS), nitric oxide (NO), and charged particles (ions) produced by surface dielectric barrier discharge (SDBD) plasma on prokaryotic (Listeria monocytogenes (Gram-positive)) and eukaryotic (human umbilical vein endothelial cells (HUVEC)) cellular function were evaluated. HUVEC and bacterial oxidative stress responses, the accumulation of nitrite in aqueous media, air ion density, and bacterial inactivation at various distances from SDBD actuators were measured. SDBD actuator designs were also varied in terms of electrode number and length to evaluate the cellular effects of plasma volume and power distribution. NO and ions were found to contribute minimally to the observed cellular effects, whereas ROS were found to cause rapid bacterial inactivation, induce eukaryotic and prokaryotic oxidative stress, and result in rapid oxidation of bovine muscle tissue. The results of this study underscore the dominance of ROS as the major plasma generated species responsible for cellular effects, with ions and RNS having a secondary, complimentary role.

Prognostic significance of receptor for hyaluronan acid-mediated motility (CD168) in acute pediatric leukemias - assessment of clinical outcome, post induction, end of treatment and minimal residual disease.

INTRODUCTION: The extracellular matrix protein hyaluronan acid plays an active in role in tumor cell proliferation and invasion. Hyaluronan acid receptors, namely CD168 or the receptor for hyaluronan acid-mediated motility (RHAMM) and CD44 have been implicated in promoting malignancy. There is a lacuna in data on the expression of the receptor in pediatric leukemias. METHODS: Pediatric patients with acute leukemia who were diagnosed, treated and followed up in our center were enrolled. The bone marrow biopsies performed prior to treatment were subjected to immunohistochemical staining (54 biopsies: acute lymphoblastic leukemia - 45, acute myeloid leukemia - 9). Blast counts were carried out at diagnosis, end of the induction phase and end of chemotherapy, the minimal residual disease was assessed and follow up details were collected. Positivity was correlated with initial blast count, post-induction blast count, minimal residual disease and patient survival. RESULTS: There was no correlation between the initial blast count and the percentage of blasts with RHAMM expression. The positive correlation between percentage of blasts expressing RHAMM and the post-induction blast count was moderate in acute myeloid leukemia (0.74) and mild in acute lymphoblastic leukemia (0.48). There was a statistically significant difference in RHAMM expression between the two minimal residual disease risk groups (p-value = 0.012) with a negative prognostic effect of RHAMM expression. Moreover, a negative prognostic effect of RHAMM expression was noted when patient survival was considered. CONCLUSION: This study shows that blasts in acute myeloid leukemia show more RHAMM positivity than those of acute lymphoblastic leukemia indicating the aggressive nature of this type of leukemia. In acute leukemias, patients with high percentages of RHAMM-positive blasts had more post-induction blasts, blasts in minimal residual disease and poorer prognosis.

Prognostic significance of receptor for hyaluronan acid-mediated motility (CD168) in acute pediatric leukemias - assessment of clinical outcome, post induction, end of treatment and minimal residual disease

ABSTRACT Introduction: The extracellular matrix protein hyaluronan acid plays an active in role in tumor cell proliferation and invasion. Hyaluronan acid receptors, namely CD168 or the receptor for hyaluronan acid-mediated motility (RHAMM) and CD44 have been implicated in promoting malignancy. There is a lacuna in data on the expression of the receptor in pediatric leukemias. Methods: Pediatric patients with acute leukemia who were diagnosed, treated and followed up in our center were enrolled. The bone marrow biopsies performed prior to treatment were subjected to immunohistochemical staining (54 biopsies: acute lymphoblastic leukemia - 45, acute myeloid leukemia - 9). Blast counts were carried out at diagnosis, end of the induction phase and end of chemotherapy, the minimal residual disease was assessed and follow up details were collected. Positivity was correlated with initial blast count, post-induction blast count, minimal residual disease and patient survival. Results: There was no correlation between the initial blast count and the percentage of blasts with RHAMM expression. The positive correlation between percentage of blasts expressing RHAMM and the post-induction blast count was moderate in acute myeloid leukemia (0.74) and mild in acute lymphoblastic leukemia (0.48). There was a statistically significant difference in RHAMM expression between the two minimal residual disease risk groups (p-value = 0.012) with a negative prognostic effect of RHAMM expression. Moreover, a negative prognostic effect of RHAMM expression was noted when patient survival was considered. Conclusion: This study shows that blasts in acute myeloid leukemia show more RHAMM positivity than those of acute lymphoblastic leukemia indicating the aggressive nature of this type of leukemia. In acute leukemias, patients with high percentages of RHAMM-positive blasts had more post-induction blasts, blasts in minimal residual disease and poorer prognosis.

[6]-Gingerol induces caspase-dependent apoptosis and prevents PMA-induced proliferation in colon cancer cells by inhibiting MAPK/AP-1 signaling.

We report mechanism-based evidence for the anticancer and chemopreventive efficacy of [6]-gingerol, the major active principle of the medicinal plant, Ginger (Zingiber officinale), in colon cancer cells. The compound was evaluated in two human colon cancer cell lines for its cytotoxic effect and the most sensitive cell line, SW-480, was selected for the mechanistic evaluation of its anticancer and chemopreventive efficacy. The non-toxic nature of [6]-gingerol was confirmed by viability assays on rapidly dividing normal mouse colon cells. [6]-gingerol inhibited cell proliferation and induced apoptosis as evidenced by externalization of phosphatidyl serine in SW-480, while the normal colon cells were unaffected. Sensitivity to [6]-gingerol in SW-480 cells was associated with activation of caspases 8, 9, 3 &7 and cleavage of PARP, which attests induction of apoptotic cell death. Mechanistically, [6]-gingerol down-regulated Phorbol Myristate Acetate (PMA) induced phosphorylation of ERK1/2 and JNK MAP kinases and activation of AP-1 transcription factor, but had only little effects on phosphorylation of p38 MAP kinase and activation of NF-kappa B. Additionally, it complemented the inhibitors of either ERK1/2 or JNK MAP kinase in bringing down the PMA-induced cell proliferation in SW-480 cells. We report the inhibition of ERK1/2/JNK/AP-1 pathway as a possible mechanism behind the anticancer as well as chemopreventive efficacy of [6]-gingerol against colon cancer.

Biochemical characterization of recombinant methionine aminopeptidases (MAPs) from Mycobacterium tuberculosis H37Rv.

Methionine aminopeptidase (MAP) performs the essential post-translational N-terminal methionine excision (NME) of nascent polypeptides during protein synthesis. To characterize MAP from Mycobacterium tuberculosis, two homolgues, mapA (Rv0734) and mapB (Rv2861c), were over expressed and purified as recombinant proteins in E. coli. In vitro activity assay of apo-MtbMAPs using L-Met-p-nitro anilide as substrate revealed MtbMAP A to be catalytically more efficient compared to MtbMAP B. Ni(2+) was the best activator of apo-MtbMAP A, whereas Ni(2+) and Co(2+) activated apo-MtbMAP B equally. MtbMAP B showed higher thermo-stability, but was feedback inhibited by higher concentrations of L-methionine. Aminopeptidase inhibitors like actinonin and bestatin inhibited both MtbMAPs, more prominently MtbMAP B. Among the site-directed mutants of MtbMAP B, substitution of metal-binding residue D142 completely abolished enzyme activity, whereas substitution of residues forming S1' pocket, C105S and T94C, had only moderate effects on substrate hydrolysis. Present study identified a specific insertion region in MtbMAP A sequence which differentiates it from other bacterial and eukaryotic MAPs. A deletion mutant lacking amino acids from this insertion region (MtbMAP A-∆164-176) was constructed to probe into their structural and functional role in activity and stability of MtbMAP A. The limited success in soluble expression of this deletion mutant suggests further optimizations of expression conditions or alternative bioinformatics approaches for further characterization of this deletion mutant of MtbMAP A.

Bioactives of microbes isolated from Western Ghat belt of Kerala show ß-lactamase inhibition along with wide spectrum antimicrobial activity.

The present study describes the exploitation of microbial biodiversity from Western Ghats of Kerala for screening of bioactives having ß-lactamase inhibitory activities. A total of 700 pure cultures were isolated and were screened for antibacterial activity against a ß-lactam resistant Bacillus cereus strain (PL 10) isolated from the same niche. Bioactive extracts made from 45 isolates showed inhibitory activities against PL 10, of which two strains showed inhibition of extended spectrum ß-lactamase (ESBL) producing Klebsiella ESBL1101 and three strains inhibited methicillin-resistant Staphylococcus aureus (MRSA) strain MRSA831. All these five strains showed wide spectrum antimicrobial activity against various fungi and bacteria. These five cultures were identified by 16S rRNA sequencing and biochemical tests and the preliminary characterizations of their bioactive extracts were carried out. This study suggests the potential of bioactives from two inhibitor-producer strains, NII 167 and NII 1054, for being developed as inhibitors against wide spectrum ß-lactam resistant strains.

Glycine in the conserved motif III modulates the thermostability and oxidative stress resistance of peptide deformylase in Mycobacterium tuberculosis.

Peptide deformylase (PDF) catalyses the removal of the N-formyl group from the nascent polypeptide during protein maturation. The PDF of Mycobacterium tuberculosis H37Rv (MtbPDF), overexpressed and purified from Escherichia coli, was characterized as an iron-containing enzyme with stability towards H(2) O(2) and moderate thermostability. Substitution of two conserved residues (G49 and L107) from MtbPDF with the corresponding residues found in human PDF affected its deformylase activity. Among characterized PDFs, glycine (G151) in motif III instead of conserved aspartate is characteristic of M. tuberculosis. Although the G151D mutation in MtbPDF increased its deformylase activity and thermostability, it also affected enzyme stability towards H(2) O(2) . Molecular dynamics and docking results confirmed improved substrate binding and catalysis for the G151D mutant and the study provides another possible molecular basis for the stability of MtbPDF against oxidizing agents.

Purification and biochemical characterization of methionine aminopeptidase (MetAP) from Mycobacterium smegmatis mc2155.

The methionine aminopeptidase (MetAP) catalyzes the removal of amino terminal methionine from newly synthesized polypeptide. MetAP from Mycobacterium smegmatis mc(2) 155 was purified from the culture lysate in four sequential steps to obtain a final purification fold of 22. The purified enzyme exhibited a molecular weight of approximately 37 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Activity staining was performed to detect the methionine aminopeptidase activity on native polyacrylamide gel. The enzyme was characterized biochemically, using L-methionine p-nitroanilide as substrate. The enzyme was found to have a temperature and pH optimum of 50 degrees C and 8.5, respectively, and was found to be stable at 50 degrees C with half-life more than 8 h. The enzyme activity was enhanced by Mg(2+) and Co(2+) and was inhibited by Fe(2+) and Cu(2+). The enzyme activity inhibited by EDTA is restored in presence of Mg(2+) suggesting the possible role of Mg(2+) as metal cofactor of the enzyme in vitro.