Faculty Bibliography

In a previous screen for Mycobacterium tuberculosis mutants that are hypersusceptible to reactive nitrogen intermediates (RNI), two genes associated with the M. tuberculosis proteasome were identified. One of these genes, pafA (proteasome accessory factor A), encodes a protein of unknown function. In this work, we determined that pafA is in an operon with two additional genes, pafB and pafC. In order to assess the contribution of these genes to RNI resistance, we isolated mutants with transposon insertions in pafB and pafC. In contrast to the pafA mutant, the pafB and pafC mutants were not severely sensitized to RNI, but pafB and pafC were nonetheless required for full RNI resistance. We also found that PafB and PafC interact with each other and that each is likely required for the stability of the other protein in M. tuberculosis. Finally, we show that the presence of PafA, but not PafB or PafC, regulates the steady-state levels of three proteasome substrates. Taken together, these data demonstrate that PafA, but not PafB or PafC, is critical for maintaining the steady-state levels of known proteasome substrates, whereas all three proteins appear to play a role in RNI resistance.

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

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

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

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

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

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

Invasion of the intestinal epithelium by Salmonella sp. requires a type III secretion system (TTSS) common in many bacterial pathogens. TTSS translocate effector proteins from bacteria into eukaryotic cells. These effectors manipulate cellular functions in order to benefit the pathogen. In the human and animal pathogen Salmonella typhimurium, the expression of genes encoding the secreted effector molecules Sip/Ssp ABCD, SigD, SptP and SopE requires both the AraC/XylS-like regulator InvF and the secretion chaperone SICA: In this work, an InvF binding site was identified in the promoter regions of three operons. SicA does not appear to affect InvF stability nor to bind DNA directly. However, SicA could be co-purified with InvF, suggesting that InvF and SicA interact with each other to activate transcription from the effector gene promoters. This is the first demonstration of a contact between a protein cofactor and an AraC/XylS family transcriptional regulator and, moreover, is the first direct evidence of an interaction between a transcriptional regulator and a TTSS chaperone. The regulation of effector genes described here for InvF and SicA may represent a new paradigm for regulation of virulence in a wide variety of pathogens

SigD is translocated into eucaryotic cells by a type III secretion system. In this work, evidence that the putative chaperone SigE directly interacts with SigD is presented. A bacterial two-hybrid system demonstrated that SigE can interact with itself and SigD. In addition, SigD was specifically copurified with SigE-His(6) on a nickel column

SigD and SigE (Salmonella invasion gene) are proteins needed for optimal invasion of Salmonella typhimurium into eukaryotic cells in vitro. SigD is a secreted protein and SigE is a putative chaperone required for SigD stability and/or secretion. SigD is secreted by a type III secretion apparatus encoded within a pathogenicity island on the Salmonella chromosome known as Salmonella pathogenicity island 1 (SPI1). The expression of sigDE, which is not linked to SPI1, is co-ordinately regulated with the SPI1 genes and is dependent on the transcriptional regulators SirA, HilA and InvF. These three proteins alone are unable to activate transcription from the sigD promoter in Escherichia coli, therefore it is likely that other factors are needed for expression. A screen for genes required for the expression of a sigD-lacZYA reporter fusion found a mutant with a transposon insertion in spaS, an SPI1 gene which encodes a putative inner-membrane component of the type III secretion system. The expression of a SPI1 operon encoding a putative chaperone (SicA) and several secreted proteins (Sips B, C, D and A) was also reduced in this mutant. The regulation defect of the spaS mutant was complemented by sicA and not by spaS. Because sicA is encoded immediately downstream of spaS, the mutation in spaS was likely to be polar on the expression of sicA. In addition, a sicA disruption mutant was as defective as an invF deletion mutant for the expression of sigD, sicA and sipC reporter fusions. The introduction of plasmids encoding invF and sicA into a non-pathogenic E. coli K-12 strain stimulated the transcription of both a sicA- and a sigD-lacZYA promoter fusion. This result suggests that InvF and SicA are sufficient for the expression of these genes. This is the first demonstration of a positive regulatory role for a putative type III secretion system chaperone in the expression of virulence genes