The ESX-1 secretion system plays a critical role in the virulence of M. tuberculosis and M. marinum , but the precise molecular and cellular mechanisms are not clearly defined. Virulent M. marinum is able to escape from the Mycobacterium -containing vacuole (MCV) into the host cell cytosol, polymerize actin, and spread from cell to cell. In this study, we have examined nine M. marinum ESX-1 mutants and the wild type by using fluorescence and electron microscopy detecting MCV membranes and actin polymerization. We conclude that ESX-1 plays an essential role in M. marinum escape from the MCV. We also show that the ESX-1 mutants acquire the ability to polymerize actin after being artificially delivered into the macrophage cytosol by hypotonic shock treatment, indicating that ESX-1 is not directly involved in initiation of actin polymerization. We provide evidence that M. marinum induces membrane pores ∼4.5 nm in diameter, and this activity correlates with ESAT-6 secretion. Importantly, purified ESAT-6, but not the other ESX-1-secreted proteins, is able to cause dose-dependent pore formation in host cell membranes. These results suggest that ESAT-6 secreted by M. marinum ESX-1 could play a direct role in producing pores in MCV membranes, facilitating M. marinum escape from the vacuole and cell-to-cell spread. Our study provides new insight into the mechanism by which ESX-1 secretion and ESAT-6 enhance the virulence of mycobacterial infection.

H-NS and Lsr2 are nucleoid-associated proteins from Gram-negative bacteria and Mycobacteria , respectively, that play an important role in the silencing of horizontally acquired foreign DNA that is more AT-rich than the resident genome. Despite the fact that Lsr2 and H-NS proteins are dissimilar in sequence and structure, they serve apparently similar functions and can functionally complement one another. The mechanism by which these xenogeneic silencers selectively target AT-rich DNA has been enigmatic. We performed high-resolution protein binding microarray analysis to simultaneously assess the binding preference of H-NS and Lsr2 for all possible 8-base sequences. Concurrently, we performed a detailed structure-function relationship analysis of their C-terminal DNA binding domains by NMR. Unexpectedly, we found that H-NS and Lsr2 use a common DNA binding mechanism where a short loop containing a “Q/RGR” motif selectively interacts with the DNA minor groove, where the highest affinity is for AT-rich sequences that lack A-tracts. Mutations of the Q/RGR motif abolished DNA binding activity. Netropsin, a DNA minor groove-binding molecule effectively outcompeted H-NS and Lsr2 for binding to AT-rich sequences. These results provide a unified molecular mechanism to explain findings related to xenogeneic silencing proteins, including their lack of apparent sequence specificity but preference for AT-rich sequences. Our findings also suggest that structural information contained within the DNA minor groove is deciphered by xenogeneic silencing proteins to distinguish genetic material that is self from nonself.

Bacterial nucleoid-associated proteins play important roles in chromosome organization and global gene regulation. We find that Lsr2 ofMycobacterium tuberculosisis a unique nucleoid-associated protein that binds AT-rich regions of the genome, including genomic islands acquired by horizontal gene transfer and regions encoding major virulence factors, such as the ESX secretion systems, the lipid virulence factors PDIM and PGL, and the PE/PPE families of antigenic proteins. Comparison of genome-wide binding data with expression data indicates that Lsr2 binding results in transcriptional repression. Domain-swapping experiments demonstrate that Lsr2 has an N-terminal dimerization domain and a C-terminal DNA-binding domain. Nuclear magnetic resonance analysis of the DNA-binding domain of Lsr2 and its interaction with DNA reveals a unique structure and a unique mechanism that enables Lsr2 to discriminately target AT-rich sequences through interactions with the minor groove of DNA. Taken together, we provide evidence that mycobacteria have employed a structurally distinct molecule with an apparently different DNA recognition mechanism to achieve a function similar to the Enterobacteriaceae H-NS, likely coordinating global gene regulation and virulence in this group of medically important bacteria.

The low permeability of the mycobacterial cell wall is thought to contribute to the well known resistance of mycobacteria to antibiotics and chemotherapeutic agents. We have used differential scanning calorimetry to demonstrate that the high temperature phase transition observed in purified cell walls, usually in the 60-70°C range, suggestive of a lipid environment of extremely low fluidity, can also be observed in whole organisms and in cell walls from which much of the free lipids was removed by extraction with Triton X-114. A survey of seven mycobacterial species demonstrated that this high temperature transition was a general property of these organisms. Cell walls isolated from two Corynebacterium species, which contain much shorter corynemycolic acids, displayed a much lower temperature transition, suggesting that the transition temperature was directly correlated to the length of mycolic acid. Methyl esters of mycolic acids were found to have a phase transition temperature that was linearly related to the amount of trans-mycolate. Both Mycobacterium avium and M. smegmatis responded to increasing growth temperature by increasing the proportion of trans-mycolate and displaying a correspondingly higher melting temperature. Whole cells of M. smegmatis grown at higher temperature allowed a less rapid influx of two lipophilic agents, norfloxacin and chenodeoxycholate. These results provide strong evidence that the nature of mycolic acid plays a crucial role in determining the fluidity and permeability of mycobacterial cell wall.