Pathogenomic analyses of Mycobacterium microti, an ESX-1-deleted member of the Mycobacterium tuberculosis complex causing disease in various hosts

Abstract

Mycobacterium microti is an animal-adapted member of the Mycobacterium tuberculosis complex (MTBC), which was originally isolated from voles, but has more recently also been isolated from other selected mammalian hosts, including occasionally from humans. Here, we have generated and analysed the complete genome sequences of five representative vole and clinical M. microti isolates using PacBio- and Illumina-based technologies, and have tested their virulence and vaccine potential in SCID (severe combined immune deficient) mouse and/or guinea pig infection models. We show that the clinical isolates studied here cluster separately in the phylogenetic tree from vole isolates and other clades from publicly available M. microti genome sequences. These data also confirm that the vole and clinical M. microti isolates were all lacking the specific RD1mic region, which in other tubercle bacilli encodes the ESX-1 type VII secretion system. Biochemical analysis further revealed marked phenotypic differences between isolates in type VII-mediated secretion of selected PE and PPE proteins, which in part were attributed to specific genetic polymorphisms. Infection experiments in the highly susceptible SCID mouse model showed that the clinical isolates were significantly more virulent than the tested vole isolates, but still much less virulent than the M. tuberculosis H37Rv control strain. The strong attenuation of the ATCC 35872 vole isolate in immunocompromised mice, even compared to the attenuated BCG (bacillus Calmette–Guérin) vaccine, and its historic use in human vaccine trials encouraged us to test this strain’s vaccine potential in a guinea pig model, where it demonstrated similar protective efficacy as a BCG control, making it a strong candidate for vaccination of immunocompromised individuals in whom BCG vaccination is contra-indicated. Overall, we provide new insights into the genomic and phenotypic variabilities and particularities of members of an understudied clade of the MTBC, which all share a recent common ancestor that is characterized by the deletion of the RD1mic region.

ESX-1-Independent Horizontal Gene Transfer by Mycobacterium tuberculosis Complex Strains

“…The results also show that M. tuberculosis and M. bovis strains, representing the globally most widely distributed pathogens among the MTBC members, apparently lack the ability to act as recipients, whereas they can still act as donors. This scenario is in agreement with the stable clonal phylogeographic lineages that are formed within the MTBC, in which the vertical transfer of genes, mutations, and deletions defines the genotypes of the daughter generations and where loss-of-function mutations cannot be repaired by HGT (25,27,54,55).…”

“…Complete read pairs were then de novo assembled using SPAdes v3.10.1 (85) (parameters --careful, -k 21,33,55 for HiSeq reads or -k 21,33,55,77 for NextSeq reads, and --phred-offset 33). The contigs thus generated were finally organized using MeDuSa v1.6 (86) (default parameters) and compared to the corresponding reference genome of each donor or recipient strain (see Table S4 in the supplemental material for details) (25,28,32,45,47,(87)(88)(89).…”

“…Interestingly, genetic determinants that define whether an M. smegmatis strain is able to act as a donor or as a recipient strain were mapped to a six-gene mating identity (mid) segment in the 39 region of the esx-1 locus, the replacement of which was linked to a switch from a recipient phenotype to a donor phenotype (9). In tuberculosis-causing mycobacteria, the ESX-1 secretion system is known to be a key virulence determinant, with its absence causing severe attenuation, as seen in the ESX-1-deleted vaccine strains Mycobacterium bovis bacillus Calmette-Guérin (BCG) (23,24) and Mycobacterium microti MP Prague (25).…”

Breaching the phagosome, the case of the tuberculosis agent

“…While the genome of Mtb (Cole et al, 1998) encodes five ESX T7SS (ESX-1 to ESX-5), in the current review, we will mainly focus on the ESX-1 system, which is absent from live attenuated vaccine strains of Mycobacterium bovis BCG (BCG) (Hsu et al, 2003;Pym, Brodin, Brosch, Huerre, & Cole, 2002) and Mycobacterium microti M.P. Prague (Orgeur et al, 2021), and its role in processes leading to induction of phagosomal rupture. The most well-known ESX-1 substrate in this context is EsxA (also known as Early Secretory Antigenic Target of 6 kDa, ESAT-6) Sorensen, Nagai, Houen, Andersen, & Andersen, 1995), which is required for full virulence of Mtb and the related fish pathogen Mycobacterium marinum (reviewed in [Groschel et al, 2016] and [Chirakos et al, 2020], respectively).…”

DataSheet_1.pdf

“…Recently, it has been shown that the ppe38-71 operon, specifically the PPE38 protein, is involved in mediating secretion of both these important subfamilies and when deleted detectable secretion is abolished (13). More recent follow up studies conducted in M. africanum and M. microti demonstrated a lack of PE-PGRS secretion in the presence of an intact ppe38-71 operon and ESX-5 secretion system (14,15). While it is clear that the ppe38-ppe71 operon is involved in PE-PGRS secretion, this phenotype can be present in other mycobacteria, driven by an independent and currently unknown mechanism.…”