Evolutionary Genomics of Cystic Fibrosis and Nosocomial Pathogens of the Mycobacterium abscessus species complex

The $\textit{Mycobacterium abscessus}$ species complex (MABSC) consists of three subspecies, $\textit{M. a. abscessus}$, $\textit{M. a. massiliense}$ and $\textit{M. a. bolletii}$. All three of these subspecies are capable of causing opportunistic pulmonary and skin and soft tissue infections in immunocompromised individuals. Infections caused by the MABSC are particularly serious in people with underlying lung conditions such as Cystic Fibrosis (CF) as these organisms are highly antibiotic resistant and the currently available treatment is toxic. This has resulted in treatment of MABSC infections failing in up to 50% of cases. Given the increasing prevalence globally of infections caused by the MABSC, particularly in individuals with CF, there is increased urgency to improve the treatment available for MABSC infections. The overall aim of this project was to use genomic analyses to increase our understanding of how the MABSC has evolved to become an opportunistic pathogen, which in turn could potentially uncover promising targets for the development of novel antibiotics. Genomic analysis of the MABSC has already shown that lineages of the MABSC are capable of indirect person to person transmission and the extent to which transmission contributed to the increasing prevalence of MABSC in people with CF became evident when the MABSC global population structure was determined. This showed that 70% of the MABSC isolates from people with CF were attributed to lineages made up of densely clustered isolates, where acquisition via indirect person-to-person transmission, as opposed to from the environment, was more likely. This thesis used population genomic approaches to i) investigate the genetic factors that drove the emergence of the most prevalent MABSC lineages, ii) look for evidence of convergence after the clonal expansion of these lineages to understand how the MABSC was continuing to adapt and spread amongst people with CF and iii) to examine the within host evolution of these pathogens to uncover how these environmental organisms were adapting to the CF lung. This thesis also used whole genome sequencing to explore the largest known outbreak of MABSC infections, a post-surgical wound infection epidemic in Brazil. Through this research the emergence of the most prevalent MABSC lineages were found to be driven by increased opportunity, probably due to the increased number of people with CF surviving longer, as opposed to the acquisition of a common genetic determinant. Not enough signal was detected after the clonal expansion of the most prevalent MABSC lineages to come to strong conclusions about genetic factors driving their continuing expansion, but strong evidence of convergent evolution was detected between MABSC isolates evolving over time within the host. The MABSC was shown to be potentially using a similar central regulatory network in response to environmental cues from the phagosome to that of $\textit{M. tuberculosis}$. The investigation into the epidemic of post-surgical wound infections in Brazil showed that a single $\textit{M. a. massiliense}$ lineage was introduced into Brazil just prior to the initial outbreak and that this lineage subsequently spread, through several waves of transmission, to multiple cities in geographically distant areas of Brazil. This highlighted how the MABSC was capable of long distance transmission and emphasised the potential of the MABSC as a nosocomial pathogen capable of causing large scale outbreaks

Average nucleotide identity of genome sequences supports the description of Rhizobium lentis sp. nov., Rhizobium bangladeshense sp. nov. and Rhizobium binae sp. nov. from lentil (Lens culinaris) nodules

Rhizobial strains isolated from effective root nodules of field-grown lentil (Lens culinaris) from different parts of Bangladesh were previously analysed using sequences of the 16S rRNA gene, three housekeeping genes (recA, atpD and glnll) and three nodulation genes (nodA, nodC and nodD), DNA fingerprinting and phenotypic characterization. Analysis of housekeeping gene sequences and DNA fingerprints indicated that the strains belonged to three novel clades in the genus Rhizobium. In present study, a representative strain from each clade was further characterized by determination of cellular fatty acid compositions, carbon substrate utilization patterns and DNA DNA hybridization and average nucleotide identity (ANI) analyses from whole-genome sequences. DNA DNA hybridization showed 50-62 % relatedness to their closest relatives (the type strains of Rhizobium etli and Rhizobium phaseoh) and 50-60 % relatedness to each other. These results were further supported by ANI values, based on genome sequencing, which were 87-92 % with their close relatives and 88-89 % with each other. On the basis of these results, three novel species, Rhizobium lentis sp. nov. (type strain BLR27(T)=LMG 28441(T)=DSM 29286(T)), Rhizobium bangladeshense sp. nov. (type strain BLR175(T)=LMG 28442(T)=DSM 29287(T)) and Rhizobium binae sp. nov. (type strain BLR195(T)=LMG 28443(T)=DSM 29288(T)), are proposed. These species share common nodulation genes (nodA, nodC and nodD) that are similar to those of the symbiovar viciae

Cell Surface Remodeling of Mycobacterium abscessus under Cystic Fibrosis Airway Growth Conditions.

Understanding the physiological processes underlying the ability of Mycobacterium abscessus to become a chronic pathogen of the cystic fibrosis (CF) lung is important to the development of prophylactic and therapeutic strategies to better control and treat pulmonary infections caused by these bacteria. Gene expression profiling of a diversity of M. abscessus complex isolates points to amino acids being significant sources of carbon and energy for M. abscessus in both CF sputum and synthetic CF medium and to the bacterium undergoing an important metabolic reprogramming in order to adapt to this particular nutritional environment. Cell envelope analyses conducted on the same representative isolates further revealed unexpected structural alterations in major cell surface glycolipids known as the glycopeptidolipids (GPLs). Besides showing an increase in triglycosylated forms of these lipids, CF sputum- and synthetic CF medium-grown isolates presented as yet unknown forms of GPLs representing as much as 10% to 20% of the total GPL content of the cells, in which the classical amino alcohol located at the carboxy terminal of the peptide, alaninol, is replaced with the branched-chain amino alcohol leucinol. Importantly, both these lipid changes were exacerbated by the presence of mucin in the culture medium. Collectively, our results reveal potential new drug targets against M. abscessus in the CF airway and point to mucin as an important host signal modulating the cell surface composition of this pathogen

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

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

Mutations in the MAB_2299c TetR Regulator Confer Cross-Resistance to Clofazimine and Bedaquiline in Mycobacterium abscessus.

New therapeutic approaches are needed against Mycobacterium abscessus, a respiratory mycobacterial pathogen that evades efforts to successfully treat infected patients. Clofazimine and bedaquiline, two drugs used for the treatment of multidrug-resistant tuberculosis, are being considered as alternatives for the treatment of lung diseases caused by M. abscessus With the aim to understand the mechanism of action of these agents in M. abscessus, we sought herein to determine the means by which M. abscessus can develop resistance. Spontaneous resistant strains selected on clofazimine, followed by whole-genome sequencing, identified mutations in MAB_2299c, encoding a putative TetR transcriptional regulator. Unexpectedly, mutants with these mutations were also cross-resistant to bedaquiline. MAB_2299c was found to bind to its target DNA, located upstream of the divergently oriented MAB_2300-MAB_2301 gene cluster, encoding MmpS/MmpL membrane proteins. Point mutations or deletion of MAB_2299c was associated with the concomitant upregulation of the mmpS and mmpL transcripts and accounted for this cross-resistance. Strikingly, deletion of MAB_2300 and MAB_2301 in the MAB_2299c mutant strain restored susceptibility to bedaquiline and clofazimine. Overall, these results expand our knowledge with respect to the regulatory mechanisms of the MmpL family of proteins and a novel mechanism of drug resistance in this difficult-to-treat respiratory mycobacterial pathogen. Therefore, MAB_2299c may represent an important marker of resistance to be considered in the treatment of M. abscessus diseases with clofazimine and bedaquiline in clinical settings

Mutations in the MAB_2299c TetR Regulator Confer Cross-Resistance to Clofazimine and Bedaquiline in Mycobacterium abscessus

International audienc

Mycobacterium abscessus pathogenesis identified by phenogenomic analyses.

Funder: Fondation BotnarFunder: European Molecular Biology OrganizationFunder: Cystic Fibrosis TrustThe medical and scientific response to emerging and established pathogens is often severely hampered by ignorance of the genetic determinants of virulence, drug resistance and clinical outcomes that could be used to identify therapeutic drug targets and forecast patient trajectories. Taking the newly emergent multidrug-resistant bacteria Mycobacterium abscessus as an example, we show that combining high-dimensional phenotyping with whole-genome sequencing in a phenogenomic analysis can rapidly reveal actionable systems-level insights into bacterial pathobiology. Through phenotyping of 331 clinical isolates, we discovered three distinct clusters of isolates, each with different virulence traits and associated with a different clinical outcome. We combined genome-wide association studies with proteome-wide computational structural modelling to define likely causal variants, and employed direct coupling analysis to identify co-evolving, and therefore potentially epistatic, gene networks. We then used in vivo CRISPR-based silencing to validate our findings and discover clinically relevant M. abscessus virulence factors including a secretion system, thus illustrating how phenogenomics can reveal critical pathways within emerging pathogenic bacteria