Functional Characterization of Nucleoid Associated Proteins Acting as Global Transcription Factors in Mycobacterium tuberculosis

The fatal lung disease tuberculosis is caused by the airborne Mycobacterium tuberculosis, a versatile pathogen adapted to rapidly changing environments. Instead of being eradicated by phagocytic cells of its human host, bacilli tune macrophages to support their own growth and even mask their presence from the immune system for several decades. Rapid adjustment of gene expression is critical for bacterial survival and heavily relies on nucleoid-associated proteins (NAPs). NAPs contribute to active DNA management by altering the chromosomal topology through bending, bridging and looping the DNA. These conformational changes can bring distant genetic loci into close spatial proximity or influence DNA supercoiling and therefore accessibility of the transcription machinery. Apart from their architectural role, NAPs moreover act as global transcription factors by direct regulation of numerous genes. In M. tuberculosis, five proteins were assigned a role as NAP. Among these, EspR, HupB and Lsr2 are crucial not only for virulence but also for cellular metabolism. This thesis focuses on the NAPs mIHF and H-NS with the objective of determining their function and target regulon. Both proteins were investigated by means of genetic manipulation, phenotype assessment and structural studies, and the efficiency of a potential new anti-tuberculosis drug acting on Lsr2 was assessed. Chrysomycin, described as specific inhibitor of Lsr2-DNA complex formation, was found to intercalate into the DNA. The resulting toxic effect on both its target M. tuberculosis as well as on eukaryotic cells rendered further development of the compound as an anti-tuberculosis drug futile. We demonstrated that Rv3852, formerly annotated as H-NS, does not act as a NAP. Deletion of the rv3852 gene had no effect on the in vitro phenotype of M. tuberculosis, did not alter nucleoid spread nor position and had no influence on virulence in mice. The mIHF protein on the other hand is not only essential for active bacterial growth, but also indispensable for survival. Generation of a conditional knockdown mutant showed that depletion of mIHF led to elongated cells devoid of septa with abnormal DNA localization and finally to cell death. The target regulon of mIHF was thoroughly studied by mapping its binding sites on the bacterial genome and by identifying genes that were differentially expressed upon depletion of the protein. We found that mIHF has a strong effect on virulence gene expression and, similar to EspR, possesses a major binding site upstream of one of the main virulence factor operons espACD. Analysis of the transcriptional response revealed that mIHF is further involved in the bacterial response to the hostâs immune system, including control of nutrient pathways as well as global protein and nucleic acid synthesis. To define how mIHF interacts with DNA and influences its 3D organisation, the protein structure of mIHF was determined by nuclear magnetic resonance spectroscopy. Binding of mIHF introduced left-hand loops into linear as well as supercoiled DNA substrates, therefore unwinding condensed DNA. We identified two DNA binding domains in mIHF and showed that its stability increased substantially upon DNA binding. All together, the findings of this thesis contribute to a better understanding of the complex gene regulatory network of M. tuberculosis, advancing the knowledge necessary to eventually defeat tuberculosis, a disease that has plagued humanity for millennia.

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

Andrej Benjak, Stewart Cole, Gaëlle Séraphine Kolly, Andréanne Lupien, Nina Theres Odermatt, Claudia Sala, Anthony Vocat

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 Delta rv3852 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 Delta rv3852 strain displayed no growth defect or morphological abnormalities. Quantitative measurement of nucleoid localization in the Delta rv3852 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.

Amer Soc Microbiology2017

Towards anti-virulence drugs targeting ESX-1 mediated pathogenesis of Mycobacterium tuberculosis

Benjamin Blasco, Jeffrey Chen, Stewart Cole, Florence Pojer

Mycobacterium tuberculosis (M. tb) the causative agent of tuberculosis (TB) causes 1.7 million deaths annually. According to the World Health Organization, in some areas of the world, 25% of TB cases are due to multi- or extensively drug resistant M. tb strains. In 2008, it was estimated that 440,000 individuals worldwide had contracted multi-drug-resistant TB with a 30% mortality rate (http://www.who.int/en/). These appalling statistics highlight the urgent need for new drugs to treat TB. Here, we propose an alternative way to fight M. tb infection by targeting key protein secretion systems required for virulence. With such an approach, rather than killing bacteria, as is the case with most antibiotics, the pathogen's ability to cause disease is diminished allowing its elimination by the immune system. We specifically focus on the specialised ESX-1 type VII secretion system (T7SS), the major and most studied virulence mechanism in M. tb (Fig. 1) that represents a new avenue for anti-TB drug development.

Elsevier2010

Spontaneous phthiocerol dimycocerosate-deficient variants of Mycobacterium tuberculosis are susceptible to gamma interferon-mediated immunity

John McKinney, Anna De Graff Tischler, Swapna Uplekar

Onset of the adaptive immune response in mice infected with Mycobacterium tuberculosis is accompanied by slowing of bacterial replication and establishment of a chronic infection. Stabilization of bacterial numbers during the chronic phase of infection is dependent on the activity of the gamma interferon (IFN-γ)-inducible nitric oxide synthase (NOS2). Previously, we described a differential signature-tagged mutagenesis screen designed to identify M. tuberculosis "counterimmune" mechanisms and reported the isolation of three mutants in the H37Rv strain background containing transposon insertions in the rv0072, rv0405, and rv2958c genes. These mutants were impaired for replication and virulence in NOS2(-/-) mice but were growth-proficient and virulent in IFN-γ(-/-) mice, suggesting that the disrupted genes were required for bacterial resistance to an IFN-γ-dependent immune mechanism other than NOS2. Here, we report that the attenuation of these strains is attributable to an underlying transposon-independent deficiency in biosynthesis of phthiocerol dimycocerosate (PDIM), a cell wall lipid that is required for full virulence in mice. We performed whole-genome resequencing of a PDIM-deficient clone and identified a spontaneous point mutation in the putative polyketide synthase PpsD that results in a G44C amino acid substitution. We demonstrate by complementation with the wild-type ppsD gene and reversion of the ppsD gene to the wild-type sequence that the ppsD(G44C) point mutation is responsible for PDIM deficiency, virulence attenuation in NOS2(-/-) and wild-type C57BL/6 mice, and a growth advantage in vitro in liquid culture. We conclude that PDIM biosynthesis is required for M. tuberculosis resistance to an IFN-γ-mediated immune response that is independent of NOS2.

2011

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

Andrej Benjak, Stewart Cole, Gaëlle Séraphine Kolly, Andréanne Lupien, Nina Theres Odermatt, Claudia Sala, Anthony Vocat

Amer Soc Microbiology2017