Ultra-low Dose Aerosol Infection of Mice with Mycobacterium tuberculosis More Closely Models Human Tuberculosis.

Tuberculosis (TB) is a heterogeneous disease manifesting in a subset of individuals infected with aerosolized Mycobacterium tuberculosis (Mtb). Unlike human TB, murine infection results in uniformly high lung bacterial burdens and poorly organized granulomas. To develop a TB model that more closely resembles human disease, we infected mice with an ultra-low dose (ULD) of between 1-3 founding bacteria, reflecting a physiologic inoculum. ULD-infected mice exhibited highly heterogeneous bacterial burdens, well-circumscribed granulomas that shared features with human granulomas, and prolonged Mtb containment with unilateral pulmonary infection in some mice. We identified blood RNA signatures in mice infected with an ULD or a conventional Mtb dose (50-100 CFU) that correlated with lung bacterial burdens and predicted Mtb infection outcomes across species, including risk of progression to active TB in humans. Overall, these findings highlight the potential of the murine TB model and show that ULD infection recapitulates key features of human TB.

Characterization of a Clp protease gene regulator and the reaeration response in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) enters a non-replicating state when exposed to low oxygen tension, a condition the bacillus encounters in granulomas during infection. Determining how mycobacteria enter and maintain this state is a major focus of research. However, from a public health standpoint the importance of latent TB is its ability to reactivate. The mechanism by which mycobacteria return to a replicating state upon re-exposure to favorable conditions is not understood. In this study, we utilized reaeration from a defined hypoxia model to characterize the adaptive response of MTB following a return to favorable growth conditions. Global transcriptional analysis identified the approximately 100 gene Reaeration Response, induced relative to both log-phase and hypoxic MTB. This response includes chaperones and proteases, as well as the transcription factor Rv2745c, which we characterize as a Clp protease gene regulator (ClgR) orthologue. During reaeration, genes repressed during hypoxia are also upregulated in a wave of transcription that includes genes crucial to transcription, translation and oxidative phosphorylation and culminates in bacterial replication. In sum, this study defines a new transcriptional response of MTB with potential relevance to disease, and implicates ClgR as a regulator involved in resumption of replication following hypoxia.

The Candida albicans mating type like locus [MTL] is not involved in chlamydospore formation.

Candida albicans produces chlamydospores, which can be used as a diagnostic tool for species identification. It has been suggested that these chlamydospores are degenerate spores. If so, then their production might be linked to the mating loci, and clinical strains that are homozygous for the C. albicans mating locus MTL may be altered in chlamydospore formation, which could cause problems in diagnostics and species identification. In Saccharomyces cerevisiae diploid cells, the heterodimeric transcriptional repressor formed by the products of the mating genes MATa1 and MATalpha2 is an important regulator of sporulation. It was therefore of interest to determine if the disruptions of the MATa1 and MATalpha2 homologs in C. albicans, MTLa1 and MTLalpha2, result in inhibition of chlamydospore formation. Laboratory strains containing disruptions of either the entire MTL locus or specific genes within the locus were assayed for their ability to form chlamydospores. Clinical strains that are homozygous for one of the two MTL loci were also assayed. No change in chlamydospore formation was seen in these strains compared to the standard laboratory strain.

Homozygosity at the Candida albicans MTL locus associated with azole resistance.

Antifungal drug resistance in the pathogenic fungus Candida albicans is a serious threat to the growing population of immunocompromised patients. This study describes a significant correlation between loss of heterozygosity at the C. albicans mating-type-like (MTL) locus and resistance to azole antifungals. A pool of 96 clinical isolates consisting of 50 azole-resistant or susceptible dose-dependent isolates and 46 azole-susceptible isolates was screened by PCR for the presence of MTLa1 and MTLalpha1. These genes were used as markers for the MTLa and MTLalpha loci. Both loci were present in 84 of the isolates. Six isolates failed to amplify MTLa1 and six failed to amplify MTLalpha1. Further PCR analysis demonstrated that loss of the MTLa1 and MTLalpha1 genes corresponded to loss of all of the loci-specific genes, resulting in homozygosity at the MTL locus. Southern analysis and single nucleotide polymorphism (SNP) analysis were used to determine that this loss of heterogeneity was due to replacement of one of the MTL loci with a duplicate of the other locus resulting in two homozygous copies of the MTL locus. Of the 12 homozygous isolates, one isolate was sensitive to azole drugs. Statistical analysis of the data demonstrates a strong correlation between homozygosity at the MTL locus and azole resistance (P<0 small middle dot003). In a set of serial isolates, an increase in azole resistance correlated with the loss of heterozygosity at the MTL locus, lending further strength to the correlation. Gene disruptions of the MTL loci were found to have no effect on azole susceptibility.