Oxygen binding and NO scavenging properties of truncated hemoglobin, HbN, of Mycobacterium smegmatis

AbstractUnraveling of microbial genome data has indicated that two distantly related truncated hemoglobins (trHbs), HbN and HbO, might occur in many species of slow-growing pathogenic mycobacteria. Involvement of HbN in bacterial defense against NO toxicity and nitrosative stress has been proposed. A gene, encoding a putative HbN homolog with conserved features of typical trHbs, has been identified within the genome sequence of fast-growing mycobacterium, Mycobacterium smegmatis. Sequence analysis of M. smegmatis HbN indicated that it is relatively smaller in size and lacks N-terminal pre-A region, carrying 12-residue polar sequence motif that is present in HbN of M. tuberculosis. HbN encoding gene of M. smegmatis was expressed in E. coli as a 12.8kD homodimeric heme protein that binds oxygen reversibly with high affinity (P50∼0.081mm Hg) and autooxidizes faster than M. tuberculosis HbN. The circular dichroism spectra indicate that HbN of M. smegmatis and M. tuberculosis are structurally similar. Interestingly, an hmp mutant of E. coli, unable to metabolize nitric oxide, exhibited very low NO uptake activity in the presence of M. smegmatis HbN as compared to HbN of M. tuberculosis. On the basis of cellular heme content, specific nitric oxide dioxygenase (NOD) activity of M. smegmatis HbN was nearly one-third of that from M. tuberculosis. Additionally, the hmp mutant of E. coli, carrying M. smegmatis HbN, exhibited nearly 10-fold lower cell survival under nitrosative stress and nitrite derived reactive nitrogen species as compared to the isogenic strain harboring HbN of M. tuberculosis. Taken together, these results suggest that NO metabolizing activity and protection provided by M. smegmatis HbN against toxicity of NO and reactive nitrogen is significantly lower than HbN of M. tuberculosis. The lower efficiency of M. smegmatis HbN for NO detoxification as compared to M. tuberculosis HbN might be related to different level of NO exposure and nitrosative stress faced by these mycobacteria during their cellular metabolism

Perception and practices of menstruation restrictions among urban adolescent girls and women in Nepal: a cross-sectional survey

Background Menstruation, a natural biologic process is associated with restrictions and superstitious beliefs in Nepal. However, factual data on women’s perspectives on menstrual practices and restrictions are scarce. This study aimed to assess socio-cultural perceptions of menstrual restrictions among urban Nepalese women in the Kathmandu valley. Methods Using a clustered random sampling, 1342 adolescent girls and women of menstruating age (≥15 years) from three urban districts in the Kathmandu valley completed a survey related to menstrual practices and restriction. This was a cross-sectional survey study using a customized program allowing pull-down, multiple choice and open-ended questions in the Nepali language. The self-administered questionnaire consisted of 13 demographic questions and 22 questions related to menstruation, menstrual hygiene, socio-cultural taboos, beliefs and practices. Univariate descriptive statistics were reported. Unadjusted associations of socio-cultural practices with ethnicity, education, four major social classes, three major religions, marital status and family type were assessed using logistic regression models. Results More than half (59%) of the participants were aged between 15- < 25 years. The majority were Hindus (84.5%), reported not praying during menstruation (83.1%) and were encouraged by their mothers (72.1%) to practice a range of menstrual restrictions. Purifying either the kitchen, bed, bedsheets or other household things on the fourth day of menstruation was reported by 66.1% of the participants, and 45.4% saw menstruation as a “bother” or “curse.” There were differences among social classes, where participants of the Janajati caste, an indigenous group, were more likely to enter places of worship [OR (95%CI): 1.74 (1.06–2.86)] and pray [OR (95%CI): 1.79 (1.18–2.71)] while menstruating, compared to the Brahmins. Participants with a master’s degree were more likely to pray while menstruating, compared to participants with less than a high school education [OR (95%CI): 2.83 (1.61–4.96)]. Conclusion This study throws light on existing social discriminations, deep-rooted cultural and religious superstitions among women, and gender inequalities in the urban areas of Kathmandu valley in Nepal. Targeted education and awareness are needed to make changes and balance between cultural and social practices during menstruation

Response of Methicillin-Resistant Staphylococcus aureus to Amicoumacin A

Amicoumacin A exhibits strong antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), hence we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in transcription of genes specifying several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which is induced in cells undergoing a collapse of Δψ. Consistent with the notion that LrgA modulates murein hydrolase activity, COL grown in the presence of amicoumacin A showed reduced autolysis, which was primarily caused by lower hydrolase activity. To gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by a serial passage method was carried out. Single point mutations generating codon substitutions were uncovered in ksgA (encoding RNA dimethyltransferase), fusA (elongation factor G), dnaG (primase), lacD (tagatose 1,6-bisphosphate aldolase), and SACOL0611 (a putative glycosyl transferase). The codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance reside in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability

Responses of Mycobacterium tuberculosis Hemoglobin Promoters to In Vitro and In Vivo Growth Conditions▿

The success of Mycobacterium tuberculosis as one of the dreaded human pathogens lies in its ability to utilize different defense mechanisms in response to the varied environmental challenges during the course of its intracellular infection, latency, and reactivation cycle. Truncated hemoglobins trHbN and trHbO are thought to play pivotal roles in the cellular metabolism of this organism during stress and hypoxia. To delineate the genetic regulation of the M. tuberculosis hemoglobins, transcriptional fusions of the promoters of the glbN and glbO genes with green fluorescent protein were constructed, and their responses were monitored in Mycobacterium smegmatis and M. tuberculosis H37Ra exposed to environmental stresses in vitro and in M. tuberculosis H37Ra after in vivo growth inside macrophages. The glbN promoter activity increased substantially during stationary phase and was nearly 3- to 3.5-fold higher than the activity of the glbO promoter, which remained more or less constant during different growth phases in M. smegmatis, as well as in M. tuberculosis H37Ra. In both mycobacterial hosts, the glbN promoter activity was induced 1.5- to 2-fold by the general nitrosative stress inducer, nitrite, as well as the NO releaser, sodium nitroprusside (SNP). The glbO promoter was more responsive to nitrite than to SNP, although the overall increase in its activity was much less than that of the glbN promoter. Additionally, the glbN promoter remained insensitive to the oxidative stress generated by H2O2, but the glbO promoter activity increased nearly 1.5-fold under similar conditions, suggesting that the trHb gene promoters are regulated differently under nitrosative and oxidative stress conditions. In contrast, transition metal-induced hypoxia enhanced the activity of both the glbN and glbO promoters at all growth phases; the glbO promoter was induced ∼2.3-fold, which was found to be the highest value for this promoter under all the conditions evaluated. Addition of iron along with nickel reversed the induction in both cases. Interestingly, a concentration-dependent decrease in the activity of both trHb gene promoters was observed when the levels of iron in the growth media were depleted by addition of an iron chelator. These results suggested that an iron/heme-containing oxygen sensor is involved in the modulation of the trHb gene promoter activities directly or indirectly in conjunction with other cellular factors. The modes of promoter regulation under different physiological conditions were found to be similar for the trHbs in both M. smegmatis and M. tuberculosis H37Ra, indicating that the promoters might be regulated by components that are common to the two systems. Confocal microscopy of THP-1 macrophages infected with M. tuberculosis carrying the trHb gene promoter fusions showed that there was a significant level of promoter activity during intracellular growth in macrophages. Time course evaluation of the promoter activity after various times up to 48 h by fluorescence-activated cell sorting analysis of the intracellular M. tuberculosis cells indicated that the glbN promoter was active at all time points assessed, whereas the activity of the glbO promoter remained at a steady-state level up to 24 h postinfection and increased ∼2-fold after 48 h of infection. Thus, the overall regulation pattern of the M. tuberculosis trHb gene promoters correlates not only with the stresses that the tubercle bacillus is likely to encounter once it is in the macrophage environment but also with our current knowledge of their functions. The in vivo studies that demonstrated for the first time expression of trHbs during macrophage infection of M. tuberculosis strongly indicate that the hemoglobins are required, and thus important, during the intracellular phase of the bacterial cycle. The present study of transcriptional regulation of M. tuberculosis hemoglobins in vitro under various stress conditions and in vivo after macrophage infection supports the hypothesis that biosynthesis of both trHbs (trHbN and trHbO) in the native host is regulated via the environmental signals that the tubercle bacillus receives during macrophage infection and growth in its human host

Identification of Conserved ABC Importers Necessary for Intracellular Survival of Legionella pneumophila in Multiple Hosts

It is established that the human pathogen Legionella pneumophila becomes significantly augmented for infection of macrophages after intracellular growth in amoebae when compared to like-strains cultivated in laboratory media. Based on this observation, we reasoned that the most critical virulence determinants of L.p. are expressed by responding to stimuli generated by the protozoan host specifically; a process we term “protozoan-priming.” We sought to identify L.p. virulence factors that were required for replication in amoebae in order to highlight the genes necessary for production of the most infectious form of the bacterium. Using a transposon mutagenesis screen, we successfully identified 12 insertions that produced bacteria severely attenuated for growth in amoebae, while retaining a functional Dot/Icm type IVb secretion system. Seven of these insertion mutants were found dispensable for growth in macrophages, revealing attractive therapeutic targets that reside upstream of the pathogen-human interface. Two candidates identified, lpg0730 and lpg0122 were required for survival and replication in amoebae and macrophage host cells. Both genes are conserved among numerous important human pathogenic bacteria that can persist or replicate in amoebae. Each gene encodes a component of an ATP binding cassette (ABC) transport complex of unknown function. We demonstrate the lpg0730 ortholog in Francisella tularensis subsp. novicida to be essential for colonization of both protozoan and mammalian host cells, highlighting conserved survival mechanisms employed by bacteria that utilize protozoa as an environmental reservoir for replication

Role of Pre-A Motif in Nitric Oxide Scavenging by Truncated Hemoglobin, HbN, of Mycobacterium tuberculosis*S⃞

Mycobacterium tuberculosis truncated hemoglobin, HbN, is endowed with a potent nitric-oxide dioxygenase activity and has been found to relieve nitrosative stress and enhance in vivo survival of a heterologous host, Salmonella enterica Typhimurium, within the macrophages. These findings implicate involvement of HbN in the defense of M. tuberculosis against nitrosative stress. The protein carries a tunnel system composed of a short and a long tunnel branch that has been proposed to facilitate diatomic ligand migration to the heme and an unusual Pre-A motif at the N terminus, which does not contribute significantly to the structural integrity of the protein, as it protrudes out of the compact globin fold. Strikingly, deletion of Pre-A region from the M. tuberculosis HbN drastically reduces its ability to scavenge nitric oxide (NO), whereas its insertion at the N terminus of Pre-A lacking HbN of Mycobacterium smegmatis improved its nitric-oxide dioxygenase activity. Titration of the oxygenated adduct of HbN and its mutants with NO indicated that the stoichiometric oxidation of protein is severalfold slower when the Pre-A region is deleted in HbN. Molecular dynamics simulations show that the excision of Pre-A motif results in distinct changes in the protein dynamics, which cause the gate of the tunnel long branch to be trapped into a closed conformation, thus impeding migration of diatomic ligands toward the heme active site. The present study, thus, unequivocally demonstrates vital function of Pre-A region in NO scavenging and unravels its unique role by which HbN might attain its efficient NO-detoxification ability

Perception and practices of menstruation restrictions among urban adolescent girls and women in Nepal: a cross-sectional survey

Abstract Background Menstruation, a natural biologic process is associated with restrictions and superstitious beliefs in Nepal. However, factual data on women’s perspectives on menstrual practices and restrictions are scarce. This study aimed to assess socio-cultural perceptions of menstrual restrictions among urban Nepalese women in the Kathmandu valley. Methods Using a clustered random sampling, 1342 adolescent girls and women of menstruating age (≥15 years) from three urban districts in the Kathmandu valley completed a survey related to menstrual practices and restriction. This was a cross-sectional survey study using a customized program allowing pull-down, multiple choice and open-ended questions in the Nepali language. The self-administered questionnaire consisted of 13 demographic questions and 22 questions related to menstruation, menstrual hygiene, socio-cultural taboos, beliefs and practices. Univariate descriptive statistics were reported. Unadjusted associations of socio-cultural practices with ethnicity, education, four major social classes, three major religions, marital status and family type were assessed using logistic regression models. Results More than half (59%) of the participants were aged between 15- < 25 years. The majority were Hindus (84.5%), reported not praying during menstruation (83.1%) and were encouraged by their mothers (72.1%) to practice a range of menstrual restrictions. Purifying either the kitchen, bed, bedsheets or other household things on the fourth day of menstruation was reported by 66.1% of the participants, and 45.4% saw menstruation as a “bother” or “curse.” There were differences among social classes, where participants of the Janajati caste, an indigenous group, were more likely to enter places of worship [OR (95%CI): 1.74 (1.06–2.86)] and pray [OR (95%CI): 1.79 (1.18–2.71)] while menstruating, compared to the Brahmins. Participants with a master’s degree were more likely to pray while menstruating, compared to participants with less than a high school education [OR (95%CI): 2.83 (1.61–4.96)]. Conclusion This study throws light on existing social discriminations, deep-rooted cultural and religious superstitions among women, and gender inequalities in the urban areas of Kathmandu valley in Nepal. Targeted education and awareness are needed to make changes and balance between cultural and social practices during menstruation.http://deepblue.lib.umich.edu/bitstream/2027.42/173732/1/12978_2020_Article_935.pd

The chemical structures of amicoumacin A and xenocoumacin 1.

<p>The chemical structures of amicoumacin A and xenocoumacin 1.</p

Northern blot analyses for SACOL0678 (A) and SACOL2176 (B) operon.

<p>Total RNA was isolated from <i>S. aureus</i> COL at 0 (t₀), 10 (t₁₀), and 40 (t₄₀) min after the addition of amicoumacin A. 10 µg of total RNA isolated from each culture was separated in a formaldehyde-agarose gel and the RNA-blotted membrane was hybridized with SACOL0678- or SACOL2173(<i>asp23</i>)-specific digoxigenin-labeled probes. The sizes of the transcripts were determined by comparison to an RNA ladder on the same gel. The corresponding stained gels are shown underneath each blot. Schematic views of the gene loci based on NCBI COL genome site are shown with predicted transcripts. σ^B indicates locations of σ^B-controlled promoters. Microarray results of each operon's genes are summarized in the right panel of the corresponding Northern blot gels. Closed squares and open squares show samples taken at t₁₀ and t₄₀, respectively. The average of triplicates and standard deviations are indicated.</p