Biomedical subjects

K Heran Darwin

Publications and source records attributed to K Heran Darwin.

6 recordsLinked to original sources

PubMed2006

Identification of substrates of the Mycobacterium tuberculosis proteasome.

The putative proteasome-associated proteins Mpa (Mycobaterium proteasomal ATPase) and PafA (proteasome accessory factor A) of the human pathogen Mycobacterium tuberculosis (Mtb) are essential for virulence and resistance to nitric oxide. However, a direct link between the proteasome protease and Mpa or PafA has never been demonstrated. Furthermore, protein degradation by bacterial proteasomes in vitro has not been accomplished, possibly due to the failure to find natural degradation substrates or other necessary proteasome co-factors. In this work, we identify the first bacterial proteasome substrates, malonyl Co-A acyl carrier protein transacylase and ketopantoate hydroxymethyltransferase, enzymes that are required for the biosynthesis of fatty acids and polyketides that are essential for the pathogenesis of Mtb. Maintenance of the physiological levels of these enzymes required Mpa and PafA in addition to proteasome protease activity. Mpa levels were also regulated in a proteasome-dependent manner. Finally, we found that a conserved tyrosine of Mpa was essential for function. Thus, these results suggest that Mpa, PafA, and the Mtb proteasome degrade bacterial proteins that are important for virulence in mice.

Acyl-Carrier Protein S-Malonyltransferase↗

PubMed2006

Self-compartmentalized bacterial proteases and pathogenesis.

Protein degradation is required for homeostasis of all living organisms. Self-compartmentalized ATP-dependent proteases are required for virulence of several pathogenic bacteria. Among the proteases implicated are ClpP and Lon, as well as the more recently identified bacterial proteasome. It is generally assumed that when a pathogen invades a host, microbial proteins become irreversibly damaged and need to be degraded. However, recent data suggest that proteolysis is also essential for virulence gene regulation. In this review, we will discuss what is known about the relationship between ATP-dependent proteolysis and pathogenesis. In addition, we will propose other potential roles these chambered proteases may have in bacterial virulence. Importantly, these proteases show promise as targets for antimicrobial therapy.

Adenosine Triphosphate↗

PubMed2005

Characterization of a Mycobacterium tuberculosis proteasomal ATPase homologue.

A screen for Mycobacterium tuberculosis (Mtb) mutants sensitive to reactive nitrogen intermediates identified transposon insertions in the presumptive proteasomal ATPase gene mpa (mycobacterium proteasome ATPase; Rv2115c). mpa mutants are attenuated in both wild type and nitric oxide synthase 2 deficient mice. In this work, we show that attenuation of mpa mutants is severe, and that Mpa is an ATPase associated with various cellular activities (AAA) ATPase that forms hexameric rings resembling the eukaryotic complex p97/valosin-containing protein (VCP). Point mutations in the conserved Walker box ATPase motifs of Mpa greatly reduced or abolished ATPase activity in vitro and abrogated protection of Mtb against acidified nitrite. A mutant Mpa protein missing only its last two amino acids retained ATPase activity, yet failed to protect Mtb against nitrite. The corresponding strain was attenuated in mice. Thus, Mpa is an ATPase whose enzymatic activity is necessary but not sufficient to protect against reactive nitrogen intermediates.

Adenosine Triphosphatases↗

PubMed2005

A glutamate-alanine-leucine (EAL) domain protein of Salmonella controls bacterial survival in mice, antioxidant defence and killing of macrophages: role of cyclic diGMP.

Signature-tagged transposon mutagenesis of Salmonella with differential recovery from wild-type and immunodeficient mice revealed that the gene here named cdgR[for c-diguanylate (c-diGMP) regulator] is required for the bacterium to resist host phagocyte oxidase in vivo. CdgR consists solely of a glutamate-alanine-leucine (EAL) domain, a predicted cyclic diGMP (c-diGMP) phosphodiesterase. Disruption of cdgR decreased bacterial resistance to hydrogen peroxide and accelerated bacterial killing of macrophages. An ultrasensitive assay revealed c-diGMP in wild-type Salmonella with increased levels in the CdgR-deficient mutant. Thus, besides its known role in regulating cellulose synthesis and biofilm formation, bacterial c-diGMP also regulates host-pathogen interactions involving antioxidant defence and cytotoxicity.

Amino Acid Sequence↗

PubMed2005

Role for nucleotide excision repair in virulence of Mycobacterium tuberculosis.

Mutations in Mycobacterium tuberculosis uvrB result in severe sensitivity to acidified nitrite, a source of nitric oxide (6). In this study, we show that a uvrB mutant is exquisitely sensitive to UV light but not to several sources of reactive oxygen species in vitro. Furthermore, a uvrB mutant was attenuated in mice as judged by an extension of life span. Attenuation in mice was partially reversed by genetic inactivation of nitric oxide synthase 2 (iNOS) and almost completely reversed in mice lacking both iNOS and phagocyte oxidase. Thus, a gene predicted to encode a key element of DNA repair is required for resistance of M. tuberculosis to both reactive nitrogen and reactive oxygen species in mice.

Animals↗

PubMed2003

The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide.

The production of nitric oxide and other reactive nitrogen intermediates (RNI) by macrophages helps to control infection by Mycobacterium tuberculosis (Mtb). However, the protection is imperfect and infection persists. To identify genes that Mtb requires to resist RNI, we screened 10,100 Mtb transposon mutants for hypersusceptibility to acidified nitrite. We found 12 mutants with insertions in seven genes representing six pathways, including the repair of DNA (uvrB) and the synthesis of a flavin cofactor (fbiC). Five mutants had insertions in proteasome-associated genes. An Mtb mutant deficient in a presumptive proteasomal adenosine triphosphatase was attenuated in mice, and exposure to proteasomal protease inhibitors markedly sensitized wild-type Mtb to RNI. Thus, the mycobacterial proteasome serves as a defense against oxidative or nitrosative stress.

Adenosine Triphosphatases↗