Highlights
- •High rates of disseminated and skeletal disease during a tuberculosis outbreak
- •Lineage 1 strains of Mycobacterium tuberculosis carry the ancestral version of EsxM
- •Ancestral, full-length EsxM alters the macrophage cytoskeleton and enhances motility
- •Ancestral EsxM promotes granuloma efflux, dissemination, and bone disease
Summary
The human pathogen Mycobacterium tuberculosis typically causes lung disease but can also disseminate to other tissues. We identified a M. tuberculosis (Mtb) outbreak presenting with unusually high rates of extrapulmonary dissemination and bone disease. We found that the causal strain carried an ancestral full-length version of the type VII-secreted effector EsxM rather than the truncated version present in other modern Mtb lineages. The ancestral EsxM variant exacerbated dissemination through enhancement of macrophage motility, increased egress of macrophages from established granulomas, and alterations in macrophage actin dynamics. Reconstitution of the ancestral version of EsxM in an attenuated modern strain of Mtb altered the migratory mode of infected macrophages, enhancing their motility. In a zebrafish model, full-length EsxM promoted bone disease. The presence of a derived nonsense variant in EsxM throughout the major Mtb lineages 2, 3, and 4 is consistent with a role for EsxM in regulating the extent of dissemination.
Introduction
Mycobacterium tuberculosis (Mtb), the etiologic agent of the disease tuberculosis (TB), is a pathogen of critical public health importance. 1 TB is predominantly a pulmonary disease, but 15%–20% of cases present with extrapulmonary manifestations.2 Extrapulmonary infections can be difficult to diagnose and treat. Skeletal TB, in turn, is a relatively uncommon manifestation of extrapulmonary TB, affecting only 148 of 7,174 (∼2%) reported TB cases in the US in 2020. 2 Although skeletal TB, or Pott’s Disease, has long been recognized, with characteristic TB-induced spinal deformities described in humans from as far back as ancient Egypt, 3 the specific bacterial factors that influence dissemination, tissue tropism, and bone disease are not well characterized.
Upon its establishment as a distinct species, Mtb evolved largely clonally. 4, 5 ,6 ,7 Although horizontal gene transfer likely contributed to its initial evolution as a human pathogen, 8 Mtb thereafter seems to have undergone relatively few horizontal gene transfer events, with some structural variants arising through genomic deletions. 7 , 9 , 10 Mtb has classically been grouped into at least seven discrete lineages. So-called “modern” lineages, which include lineages 2, 3, and 4, (L2, L3, and L4) are broadly distributed throughout the world. 9 Other lineages (L1, L5, L6, and L7) are more geographically constrained.
11 L1 strains, although geographically limited, still account for many overall cases and have been previously reported to induce distinct inflammatory phenotypes and differentially modulate innate immune signaling in the human host. 12 ,13 ,14 ,15 Pathogenic mycobacteria achieve dissemination through a remarkable ability to spread within their hosts. They hijack host macrophages as both a major replicative niche and for delivery to distal locations within and between tissues. 16 Macrophage function and motility influence dissemination of mycobacterial disease through a number of mechanisms, including macrophage survival and cell death, and efflux from initial nidi to new sites.
17, 18, 19 ,20 The dynamics of granuloma formation, dissolution, and resolution also influence the trajectory of infection and dissemination in zebrafish and macaque models.
21, 22, 23, 24 Macrophage and granuloma behavior is heavily influenced by dedicated bacterial effectors secreted through type VII secretion systems. The ESX-1 system, notably absent in the attenuated BCG vaccine strain, 25 ,26, 27, 28 plays multiple roles in virulence, most prominently in the permeabilization of the phagosomal membrane in infected macrophages.
29, 30 ,31 , 32 ,33 , 34 Mutants defective for the small secreted effectors specific to the ESX-1 secretion system—EsxA and EsxB—display altered virulence. 35 Similarly, the paralogous ESX-3 system regulates important pathogen-host interactions, including iron acquisition,36, 37 and, through interactions of the small secreted effectors EsxG and EsxH with the ESCRT complex, host membrane trafficking, and damage response.38, 39 ,40 ESX-5, the most recently evolved of the paralogous type VII secretion systems, is found only in the slow-growing pathogenic mycobacteria, including Mtb and Mycobacterium marinum.
41 ,42 ESX-5 has been implicated in secretion of Mtb’s abundant PE and PPE family proteins 43 44 and the CpnT toxin. 45
However, a biological role of any of the small secreted effectors specific to ESX-5, including EsxM and EsxN, has not fully been examined.
Here, we describe an unusual outbreak of Mtb with high rates of extrapulmonary dissemination and bone disease. We uncover a functional variant in the ESX-5 secreted effector EsxM that precisely coincides with a transition from the ancestral allele present in Mtb lineages 1, 5, 6, and 7 to a derived truncation allele in lineages L2– L4. The ancestral version of EsxM present in the outbreak strain, and generally in L1 strains, leads to alterations in the modality of infected host macrophage migration and the rate of egress of infected macrophages from granulomas. Mtb generally requires airborne lung-to-lung transmission, so we therefore propose that limiting the degree of dissemination to tissues outside the lung may be advantageous with respect to the likelihood of transmission. We infer that the stop codon in EsxM was introduced in the most recent common ancestor of the L2–L4 Mtb strains, leading to decreased rates of dissemination and skeletal disease for strains carrying this variant.
Results
A North Carolina outbreak with high rates of skeletal disease
We investigated a TB outbreak with extremely high rates of disseminated and skeletal disease (Figures 1A and 1B ). The index case, a man originally from Vietnam, was diagnosed with pulmonary TB after over a year of symptoms, and a contact investigation was carried out. 46
Seven secondary cases of active TB were identified (Figures 1A and 1B), and six of the seven (86%) presented with extrapulmonary disease. Remarkably, though the reported frequency of skeletal disease is 2% of all US cases, 2 four of the extrapulmonary cases (57% of cases in the outbreak) had skeletal disease. The binomial probability of observing four or more bone cases among seven TB patients if the probability of each case having bone TB is equal to the population proportion is approximately 5×10−6. Two of the bone TB cases had a single site of disease in the spine, and the other two had diffuse bony disease involving the ribs, scapula, iliac crest, spine, and sternum (Figures 1C–1E). One of the patients with diffuse bone disease had concurrent pulmonary involvement but no other site, and the other had no site of disease outside the bones. All secondary case patients were US-born (one White, the rest Black), HIV-negative, genetically unrelated to the index case and each other, and seemingly otherwise immunocompetent. The strains isolated from each patient had identical genotypes by mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) typing and spoligotyping, suggesting a single transmitting strain, which we refer to as NCG (Figure S1)…
