scholarly journals Rv3852 (H-NS) of Mycobacterium tuberculosis Is Not Involved in Nucleoid Compaction and Virulence Regulation

2017 ◽  
Vol 199 (16) ◽  
Author(s):  
Nina T. Odermatt ◽  
Claudia Sala ◽  
Andrej Benjak ◽  
Gaëlle S. Kolly ◽  
Anthony Vocat ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Regulatory Networks ◽  
Sequence Similarity ◽  
Growth Defect ◽  
Foreign Gene ◽  
Logarithmic Growth Phase ◽  
Content Type ◽  
Associated Proteins ◽  
Transcription Regulators ◽  
Bacterial Genes

ABSTRACT A handful of nucleoid-associated proteins (NAPs) regulate the vast majority of genes in a bacterial cell. H-NS, the histone-like nucleoid-structuring protein, is one of these NAPs and protects Escherichia coli from foreign gene expression. Though lacking any sequence similarity with E. coli H-NS, Rv3852 was annotated as the H-NS ortholog in Mycobacterium tuberculosis, as it resembles human histone H1. The role of Rv3852 was thoroughly investigated by immunoblotting, subcellular localization, construction of an unmarked rv3852 deletion in the M. tuberculosis genome, and subsequent analysis of the resulting Δrv3852 strain. We found that Rv3852 was predominantly present in the logarithmic growth phase with a decrease in protein abundance in stationary phase. Furthermore, it was strongly associated with the cell membrane and not detected in the cytosolic fraction, nor was it secreted. The Δrv3852 strain displayed no growth defect or morphological abnormalities. Quantitative measurement of nucleoid localization in the Δrv3852 mutant strain compared to that in the parental H37Rv strain showed no difference in nucleoid position or spread. Infection of macrophages as well as severe combined immunodeficient (SCID) mice demonstrated that loss of Rv3852 had no detected influence on the virulence of M. tuberculosis. We thus conclude that M. tuberculosis Rv3852 is not involved in pathogenesis and is not a typical NAP. The existence of an as yet undiscovered Rv3852 ortholog cannot be excluded, although this role is likely played by the well-characterized Lsr2 protein. IMPORTANCE Mycobacterium tuberculosis is the causative agent of the lung infection tuberculosis, claiming more than 1.5 million lives each year. To understand the mechanisms of latent infection, where M. tuberculosis can stay dormant inside the human host, we require deeper knowledge of the basic biology and of the regulatory networks. In our work, we show that Rv3852, previously annotated as H-NS, is not a typical nucleoid-associated protein (NAP) as expected from its initial annotation. Rv3852 from M. tuberculosis has neither influence on nucleoid shape or compaction nor a role in virulence. Our findings reduce the repertoire of identified nucleoid-associated proteins in M. tuberculosis to four transcription regulators and underline the importance of genetic studies to assign a function to bacterial genes.

scholarly journals Loss-of-Function Mutations in HspR Rescue the Growth Defect of a Mycobacterium tuberculosis Proteasome Accessory Factor E (pafE) Mutant

2017 ◽  
Vol 199 (7) ◽  
Author(s):  
Jordan B. Jastrab ◽  
Marie I. Samanovic ◽  
Richard Copin ◽  
Bo Shopsin ◽  
K. Heran Darwin
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Heat Shock ◽  
Protein Quality ◽  
Normal Growth ◽  
Culture Conditions ◽  
Growth Defect ◽  
Loss Of Function ◽  
Content Type ◽  
Accessory Factor

ABSTRACT Mycobacterium tuberculosis uses a proteasome to degrade proteins by both ATP-dependent and -independent pathways. While much has been learned about ATP-dependent degradation, relatively little is understood about the ATP-independent pathway, which is controlled by Mycobacterium tuberculosis proteasome accessory factor E (PafE). Recently, we found that a Mycobacterium tuberculosis pafE mutant has slowed growth in vitro and is sensitive to killing by heat stress. However, we did not know if these phenotypes were caused by an inability to degrade the PafE-proteasome substrate HspR (heat shock protein repressor), an inability to degrade any damaged or misfolded proteins, or a defect in another protein quality control pathway. To address this question, we characterized pafE suppressor mutants that grew similarly to pafE + bacteria under normal culture conditions. All but one suppressor mutant analyzed contained mutations that inactivated HspR function, demonstrating that the slowed growth and heat shock sensitivity of a pafE mutant were caused primarily by the inability of the proteasome to degrade HspR. IMPORTANCE Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required for virulence. We recently discovered a proteasome cofactor, PafE, which is required for the normal growth, heat shock resistance, and full virulence of M. tuberculosis. In this study, we demonstrate that PafE influences this phenotype primarily by promoting the expression of protein chaperone genes that are necessary for surviving proteotoxic stress.

scholarly journals The Mycobacterium tuberculosis SecA2 System Subverts Phagosome Maturation To Promote Growth in Macrophages

2012 ◽  
Vol 80 (3) ◽  
pp. 996-1006 ◽  
Author(s):  
Jonathan Tabb Sullivan ◽  
Ellen F. Young ◽  
Jessica R. McCann ◽  
Miriam Braunstein
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protein Export ◽  
Growth Defect ◽  
Phagosome Maturation ◽  
Wild Type ◽  
Intracellular Growth ◽  
Maturation Arrest ◽  
Content Type ◽  
Late Endosomes ◽  
Export System

The ability ofMycobacterium tuberculosisto grow in macrophages is critical to the virulence of this important pathogen. One wayM. tuberculosisis thought to maintain a hospitable niche in macrophages is by arresting the normal process of phagosomes maturing into acidified phagolysosomes. The process of phagosome maturation arrest byM. tuberculosisis not fully understood, and there has remained a need to firmly establish a requirement for phagosome maturation arrest forM. tuberculosisgrowth in macrophages. Other intracellular pathogens that control the phagosomal environment use specialized protein export systems to deliver effectors of phagosome trafficking to the host cell. InM. tuberculosis, the accessory SecA2 system is a specialized protein export system that is required for intracellular growth in macrophages. In studying the importance of the SecA2 system in macrophages, we discovered that SecA2 is required for phagosome maturation arrest. Shortly after infection, phagosomes containing a ΔsecA2mutant ofM. tuberculosiswere more acidified and showed greater association with markers of late endosomes than phagosomes containing wild-typeM. tuberculosis. We further showed that inhibitors of phagosome acidification rescued the intracellular growth defect of the ΔsecA2mutant, which demonstrated that the phagosome maturation arrest defect of the ΔsecA2mutant is responsible for the intracellular growth defect. This study demonstrates the importance of phagosome maturation arrest forM. tuberculosisgrowth in macrophages, and it suggests there are effectors of phagosome maturation that are exported into the host environment by the accessory SecA2 system.

scholarly journals Acquisition of Iron Is Required for Growth of Salmonella spp. in Tomato Fruit

2015 ◽  
Vol 81 (11) ◽  
pp. 3663-3670 ◽  
Author(s):  
Staci L. Nugent ◽  
Fanhong Meng ◽  
Gregory B. Martin ◽  
Craig Altier
Keyword(s):  
Iron Acquisition ◽  
Tomato Fruit ◽  
Meat Products ◽  
Growth Defect ◽  
Ferric Uptake Regulator ◽  
Wild Type ◽  
Salmonella Spp ◽  
Content Type ◽  
Food Borne ◽  
Bacterial Genes

ABSTRACTSalmonellaremains a leading cause of bacterial food-borne disease, sickening millions each year. Although outbreaks of salmonellosis have traditionally been associated with contaminated meat products, recent years have seen numerous disease cases caused by the consumption of produce. Tomatoes have been specifically implicated, due to the ability ofSalmonellaspp. to enter the tomato fruit and proliferate within, making the decontamination of the raw product impossible. To investigate the genetic means by whichSalmonellais able to survive and proliferate within tomatoes, we conducted a screen for bacterial genes ofSalmonella entericaserovar Montevideo specifically induced after inoculation into ripe tomato fruit. Among these genes, we found 17 members of the previously described anaerobic Fur (ferric uptake regulator) regulon. Fur is a transcriptional and posttranscriptional regulator known to sense iron, suggesting the importance of this mineral toSalmonellawithin tomatoes. To test whether iron acquisition is essential forSalmonellagrowth in tomatoes, we tested a ΔfepDGCmutant, which lacks the ability to import iron-associated siderophores. This mutant grew significantly more poorly within tomatoes than did the wild type, but the growth defect of the mutant was fully reversed by the addition of exogenous iron, demonstrating the need for bacterial iron scavenging. Further, dependence upon iron was not apparent forSalmonellagrowing in filtered tomato juice, implicating the cellular fraction of the fruit as an important mediator of iron acquisition by the bacteria.

scholarly journals Characterization of Large Deletion Mutants of Mycobacterium tuberculosis Selected for Isoniazid Resistance

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
Catherine Vilchèze ◽  
Rajagopalan Saranathan ◽  
Brian Weinrick ◽  
William R. Jacobs
Keyword(s):  
Oxidative Stress ◽  
Mycobacterium Tuberculosis ◽  
Large Deletion ◽  
Growth Defect ◽  
Intracellular Growth ◽  
Isoniazid Resistance ◽  
Content Type ◽  
Genomic Deletions

ABSTRACT Large genomic deletions (LGDs) (6 to 63 kbp) were observed in isoniazid-resistant Mycobacterium tuberculosis mutants derived from four M. tuberculosis strains. These LGDs had no growth defect in vitro but could be defective in intracellular growth and showed various sensitivities toward oxidative stress despite lacking katG. The LGD regions comprise 74 genes, mostly of unknown function, that may be important for M. tuberculosis intracellular growth and protection against oxidative stress.

scholarly journals Systematic bacterialization of yeast genes identifies a near-universally swappable pathway

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Aashiq H Kachroo ◽  
Jon M Laurent ◽  
Azat Akhmetov ◽  
Madelyn Szilagyi-Jones ◽  
Claire D McWhite ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Predictive Power ◽  
Common Ancestor ◽  
Sequence Similarity ◽  
Growth Defect ◽  
Biosynthesis Pathway ◽  
Mitochondrial Localization ◽  
Bacterial Genes ◽  
Yeast Genes

Eukaryotes and prokaryotes last shared a common ancestor ~2 billion years ago, and while many present-day genes in these lineages predate this divergence, the extent to which these genes still perform their ancestral functions is largely unknown. To test principles governing retention of ancient function, we asked if prokaryotic genes could replace their essential eukaryotic orthologs. We systematically replaced essential genes in yeast by their 1:1 orthologs from Escherichia coli. After accounting for mitochondrial localization and alternative start codons, 31 out of 51 bacterial genes tested (61%) could complement a lethal growth defect and replace their yeast orthologs with minimal effects on growth rate. Replaceability was determined on a pathway-by-pathway basis; codon usage, abundance, and sequence similarity contributed predictive power. The heme biosynthesis pathway was particularly amenable to inter-kingdom exchange, with each yeast enzyme replaceable by its bacterial, human, or plant ortholog, suggesting it as a near-universally swappable pathway.

scholarly journals Effect of Moxifloxacin plus Pretomanid against Mycobacterium tuberculosis in Log Phase, Acid Phase, and Nonreplicating-Persister Phase in an In Vitro Assay

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Carolina de Miranda Silva ◽  
Amirhossein Hajihosseini ◽  
Jenny Myrick ◽  
Jocelyn Nole ◽  
Arnold Louie ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Animal Studies ◽  
Screening Strategy ◽  
Infection Model ◽  
Logarithmic Growth Phase ◽  
Vitro Assay ◽  
Content Type ◽  
Successful Approach ◽  
Resistant Strains

ABSTRACT Combination therapy is a successful approach to treat tuberculosis in patients with susceptible strains of Mycobacterium tuberculosis. However, the emergence of resistant strains requires identification of new, effective therapies. Pretomanid (PA824) and moxifloxacin (MXF) are promising options currently under evaluation in clinical trials for the treatment of susceptible and resistant mycobacteria. We applied our recently described screening strategy to characterize the interaction between PA824 and MXF toward the killing of M. tuberculosis in logarithmic growth phase (log phase), acid phase, and nonreplicating-persister (NRP) phase. Respective in vitro data generated for the H37Rv and 18b strains were evaluated in a microdilution plate system containing both drugs in combination. The Universal Response Surface Approach model from Greco et al. (W. R. Greco, G. Bravo, and J. C. Parsons, Pharmacol Rev 47:331–385, 1995) was used to characterize the nature of the interaction between both drugs; synergistic or additive combinations would prompt additional evaluation in the hollow-fiber infection model (HFIM) and in animal studies. The interaction between MXF and PA824 was additive against M. tuberculosis organisms in acid phase (interaction parameter [α] = 5.56e−8 [95% confidence interval {CI} = −0.278 to 0.278] and α = 0.408 [95% CI = 0.105 to 0.711], respectively), NRP phase (α = 0.625 [95% CI = −0.556 to 1.81] and α = 2.92 [95% CI = 0.215 to 5.63], respectively), and log phase (α = 1.57e−6 [95% CI = −0.930 to 0.930] and α = 1.83e−6 [95% CI = −0.929 and 0.929], respectively), prompting further testing of this promising combination for the treatment of tuberculosis in the HFIM and in animal studies.

Rv0802c is an acyltransferase that succinylates and acetylates Mycobacterium tuberculosis nucleoid-associated protein HU

Microbiology ◽  
2021 ◽  
Vol 167 (7) ◽  
Author(s):  
Chinmay Anand ◽  
Meghna Santoshi ◽  
Prakruti R Singh ◽  
Valakunja Nagaraja
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Regulation Of Gene Expression ◽  
Cellular Functions ◽  
Mobility Shift ◽  
Post Translational Modifications ◽  
Content Type ◽  
Associated Proteins ◽  
Carboxyl Terminal ◽  
Mobility Shift Assay

Among the nucleoid-associated proteins (NAPs), HU is the most conserved in eubacteria, engaged in overall chromosome organization and regulation of gene expression. Unlike other bacteria, HU from Mycobacterium tuberculosis (MtHU), has a long carboxyl terminal domain enriched in basic amino acids, resembling eukaryotic histone N-terminal tails. As with histones, MtHU undergoes post-translational modifications and we have previously identified interacting kinases, methyltransferases, an acetyltransferase and a deacetylase. Here we show that Rv0802c interacts and succinylates MtHU. Although categorized as a succinyltransferase, we show that this GNAT superfamily member can catalyse both succinylation and acetylation of MtHU with comparable kinetic parameters. Like acetylation of MtHU, succinylation of MtHU caused reduced interaction of the NAP with DNA, determined by electrophoretic mobility shift assay and surface plasmon resonance. However, in vivo expression of Rv0802c did not significantly alter the nucleoid architecture. Although such succinylation of NAPs is rare, these modifications of the archetypal NAP may provide avenues to the organism to compensate for the underrepresentation of NAPs in its genome to control the dynamics of nucleoid architecture and cellular functions.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals Use of Proteins Identified through a Functional Genomic Screen To Develop a Protein Subunit Vaccine That Provides Significant Protection against Virulent Streptococcus suis in Pigs

2017 ◽  
Vol 86 (3) ◽  
Author(s):  
Susan L. Brockmeier ◽  
Crystal L. Loving ◽  
Tracy L. Nicholson ◽  
Jinhong Wang ◽  
Sarah E. Peters ◽  
...  
Keyword(s):  
Immune Response ◽  
Vaccine Trial ◽  
Streptococcus Suis ◽  
Protein Subunit ◽  
Content Type ◽  
Degree Of Protection ◽  
Wide Range ◽  
Associated Proteins ◽  
Clinical Protection ◽  
Cellular Immune

ABSTRACT Streptococcus suis is a bacterium that is commonly carried in the respiratory tract and that is also one of the most important invasive pathogens of swine, commonly causing meningitis, arthritis, and septicemia. Due to the existence of many serotypes and a wide range of immune evasion capabilities, efficacious vaccines are not readily available. The selection of S. suis protein candidates for inclusion in a vaccine was accomplished by identifying fitness genes through a functional genomics screen and selecting conserved predicted surface-associated proteins. Five candidate proteins were selected for evaluation in a vaccine trial and administered both intranasally and intramuscularly with one of two different adjuvant formulations. Clinical protection was evaluated by subsequent intranasal challenge with virulent S. suis . While subunit vaccination with the S. suis proteins induced IgG antibodies to each individual protein and a cellular immune response to the pool of proteins and provided substantial protection from challenge with virulent S. suis , the immune response elicited and the degree of protection were dependent on the parenteral adjuvant given. Subunit vaccination induced IgG reactive against different S. suis serotypes, indicating a potential for cross protection.

scholarly journals Multi-omic regulatory networks capture downstream effects of kinase inhibition in Mycobacterium tuberculosis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Albert T. Young ◽  
Xavier Carette ◽  
Michaela Helmel ◽  
Hanno Steen ◽  
Robert N. Husson ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mycobacterium Tuberculosis ◽  
Regulatory Network ◽  
Regulatory Networks ◽  
Protein Interaction Data ◽  
Kinase Inhibition ◽  
Protein Protein Interaction ◽  
Downstream Effects ◽  
Regulate Cell Growth ◽  
Regulatory Effects

AbstractThe ability of Mycobacterium tuberculosis (Mtb) to adapt to diverse stresses in its host environment is crucial for pathogenesis. Two essential Mtb serine/threonine protein kinases, PknA and PknB, regulate cell growth in response to environmental stimuli, but little is known about their downstream effects. By combining RNA-Seq data, following treatment with either an inhibitor of both PknA and PknB or an inactive control, with publicly available ChIP-Seq and protein–protein interaction data for transcription factors, we show that the Mtb transcription factor (TF) regulatory network propagates the effects of kinase inhibition and leads to widespread changes in regulatory programs involved in cell wall integrity, stress response, and energy production, among others. We also observe that changes in TF regulatory activity correlate with kinase-specific phosphorylation of those TFs. In addition to characterizing the downstream regulatory effects of PknA/PknB inhibition, this demonstrates the need for regulatory network approaches that can incorporate signal-driven transcription factor modifications.

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