Mechanical morphotype switching as an adaptive response in mycobacteria.

Invading microbes face a myriad of cidal mechanisms of phagocytes that inflict physical damage to microbial structures. How intracellular bacterial pathogens adapt to these stresses is not fully understood. Here, we report the discovery of a virulence mechanism by which changes to the mechanical stiffness of the mycobacterial cell surface confer refraction to killing during infection. Long-term time-lapse atomic force microscopy was used to reveal a process of "mechanical morphotype switching" in mycobacteria exposed to host intracellular stress. A "soft" mechanical morphotype switch enhances tolerance to intracellular macrophage stress, including cathelicidin. Both pharmacologic treatment, with bedaquiline, and a genetic mutant lacking uvrA modified the basal mechanical state of mycobacteria into a soft mechanical morphotype, enhancing survival in macrophages. Our study proposes microbial cell mechanical adaptation as a critical axis for surviving host-mediated stressors.

Development of Inhibitors of SAICAR Synthetase (PurC) from Mycobacterium abscessus Using a Fragment-Based Approach.

Mycobacterium abscessus (Mab) has emerged as a challenging threat to individuals with cystic fibrosis. Infections caused by this pathogen are often impossible to treat due to the intrinsic antibiotic resistance leading to lung malfunction and eventually death. Therefore, there is an urgent need to develop new drugs against novel targets in Mab to overcome drug resistance and subsequent treatment failure. In this study, SAICAR synthetase (PurC) from Mab was identified as a promising target for novel antibiotics. An in-house fragment library screen and a high-throughput X-ray crystallographic screen of diverse fragment libraries were explored to provide crucial starting points for fragment elaboration. A series of compounds developed from fragment growing and merging strategies, guided by crystallographic information and careful hit-to-lead optimization, have achieved potent nanomolar binding affinity against the enzyme. Some compounds also show a promising inhibitory effect against Mab and Mtb. This work utilizes a fragment-based design and demonstrates for the first time the potential to develop inhibitors against PurC from Mab.

Unique Features of Mycobacterium abscessus Biofilms Formed in Synthetic Cystic Fibrosis Medium.

Characterizing Mycobacterium abscessus complex (MABSC) biofilms under host-relevant conditions is essential to the design of informed therapeutic strategies targeted to this persistent, drug-tolerant, population of extracellular bacilli. Using synthetic cystic fibrosis medium (SCFM) which we previously reported to closely mimic the conditions encountered by MABSC in actual cystic fibrosis (CF) sputum and a new model of biofilm formation, we show that MABSC biofilms formed under these conditions are substantially different from previously reported biofilms grown in standard laboratory media in terms of their composition, gene expression profile and stress response. Extracellular DNA (eDNA), mannose-and glucose-containing glycans and phospholipids, rather than proteins and mycolic acids, were revealed as key extracellular matrix (ECM) constituents holding clusters of bacilli together. None of the environmental cues previously reported to impact biofilm development had any significant effect on SCFM-grown biofilms, most likely reflecting the fact that SCFM is a nutrient-rich environment in which MABSC finds a variety of ways of coping with stresses. Finally, molecular determinants were identified that may represent attractive new targets for the development of adjunct therapeutics targeting MABSC biofilms in persons with CF.

Increased Virulence of Outer Membrane Porin Mutants of Mycobacterium abscessus.

Chronic pulmonary infections caused by non-tuberculous mycobacteria of the Mycobacterium abscessus complex (MABSC) are emerging as a global health problem and pose a threat to susceptible individuals with structural lung disease such as cystic fibrosis. The molecular mechanisms underlying the pathogenicity and intrinsic resistance of MABSC to antibiotics remain largely unknown. The involvement of Msp-type porins in the virulence and biocide resistance of some rapidly growing non-tuberculous mycobacteria and the finding of deletions and rearrangements in the porin genes of serially collected MABSC isolates from cystic fibrosis patients prompted us to investigate the contribution of these major surface proteins to MABSC infection. Inactivation by allelic replacement of the each of the two Msp-type porin genes of M. abscessus subsp. massiliense CIP108297, mmpA and mmpB, led to a marked increase in the virulence and pathogenicity of both mutants in murine macrophages and infected mice. Neither of the mutants were found to be significantly more resistant to antibiotics. These results suggest that adaptation to the host environment rather than antibiotic pressure is the key driver of the emergence of porin mutants during infection.

Stepwise pathogenic evolution of Mycobacterium abscessus.

Although almost all mycobacterial species are saprophytic environmental organisms, a few, such as Mycobacterium tuberculosis, have evolved to cause transmissible human infection. By analyzing the recent emergence and spread of the environmental organism M. abscessus through the global cystic fibrosis population, we have defined key, generalizable steps involved in the pathogenic evolution of mycobacteria. We show that epigenetic modifiers, acquired through horizontal gene transfer, cause saltational increases in the pathogenic potential of specific environmental clones. Allopatric parallel evolution during chronic lung infection then promotes rapid increases in virulence through mutations in a discrete gene network; these mutations enhance growth within macrophages but impair fomite survival. As a consequence, we observe constrained pathogenic evolution while person-to-person transmission remains indirect, but postulate accelerated pathogenic adaptation once direct transmission is possible, as observed for M. tuberculosis Our findings indicate how key interventions, such as early treatment and cross-infection control, might restrict the spread of existing mycobacterial pathogens and prevent new, emergent ones.

IQG-607 abrogates the synthesis of mycolic acids and displays intracellular activity against Mycobacterium tuberculosis in infected macrophages.

In this work, the antitubercular activity of a pentacyano(isoniazid)ferrate(II) compound (IQG-607) was investigated using a macrophage model of Mycobacterium tuberculosis infection. Importantly, treatment of M.-tuberculosis-infected macrophages with IQG-607 significantly diminished the number of CFU compared with the untreated control group. The antitubercular activity of IQG-607 was similar to that observed for the positive control drugs isoniazid and rifampicin. Nevertheless, higher concentrations of IQG-607 produced a significantly greater reduction in bacterial load compared with the same concentrations of isoniazid. Analysis of the mechanism of action of IQG-607 revealed that the biosynthesis of mycolic acids was blocked. The promising activity of IQG-607 in infected macrophages and the experimental determination of its mechanism of action may help in further studies aimed at the development of a new antimycobacterial agent.