Mycobacterium tuberculosis Requires the Outer Membrane Lipid Phthiocerol Dimycocerosate for Starvation-Induced Antibiotic Tolerance.

Tolerance of Mycobacterium tuberculosis to antibiotics contributes to the long duration of tuberculosis (TB) treatment and the emergence of drug-resistant strains. M. tuberculosis drug tolerance is induced by nutrient restriction, but the genetic determinants that promote antibiotic tolerance triggered by nutrient limitation have not been comprehensively identified. Here, we show that M. tuberculosis requires production of the outer membrane lipid phthiocerol dimycocerosate (PDIM) to tolerate antibiotics under nutrient-limited conditions. We developed an arrayed transposon (Tn) mutant library in M. tuberculosis Erdman and used orthogonal pooling and transposon sequencing (Tn-seq) to map the locations of individual mutants in the library. We screened a subset of the library (~1,000 mutants) by Tn-seq and identified 32 and 102 Tn mutants with altered tolerance to antibiotics under stationary-phase and phosphate-starved conditions, respectively. Two mutants recovered from the arrayed library, ppgK::Tn and clpS::Tn, showed increased susceptibility to two different drug combinations under both nutrient-limited conditions, but their phenotypes were not complemented by the Tn-disrupted gene. Whole-genome sequencing revealed single nucleotide polymorphisms in both the ppgK::Tn and clpS::Tn mutants that prevented PDIM production. Complementation of the clpS::Tn ppsD Q291* mutant with ppsD restored PDIM production and antibiotic tolerance, demonstrating that loss of PDIM sensitized M. tuberculosis to antibiotics. Our data suggest that drugs targeting production of PDIM, a critical M. tuberculosis virulence determinant, have the potential to enhance the efficacy of existing antibiotics, thereby shortening TB treatment and limiting development of drug resistance. IMPORTANCE Mycobacterium tuberculosis causes 10 million cases of active TB disease and over 1 million deaths worldwide each year. TB treatment is complex, requiring at least 6 months of therapy with a combination of antibiotics. One factor that contributes to the length of TB treatment is M. tuberculosis phenotypic antibiotic tolerance, which allows the bacteria to survive prolonged drug exposure even in the absence of genetic mutations causing drug resistance. Here, we report a genetic screen to identify M. tuberculosis genes that promote drug tolerance during nutrient starvation. Our study revealed the outer membrane lipid phthiocerol dimycocerosate (PDIM) as a key determinant of M. tuberculosis antibiotic tolerance triggered by nutrient starvation. Our study implicates PDIM synthesis as a potential target for development of new TB drugs that would sensitize M. tuberculosis to existing antibiotics to shorten TB treatment.

Plasmodium Secretion Induces Hepatocyte Lysosome Exocytosis and Promotes Parasite Entry.

The invasion of a suitable host hepatocyte by Plasmodium sporozoites is an essential step in malaria infection. We demonstrate that in infected hepatocytes, lysosomes are redistributed away from the nucleus, and surface exposure of lysosome-associated membrane protein 1 (LAMP1) is increased. Lysosome exocytosis in infected cells occurs independently of sporozoite traversal. Instead, a sporozoite-secreted factor is sufficient for the process. Knockdown of SNARE proteins involved in lysosome-plasma membrane fusion reduces lysosome exocytosis and Plasmodium infection. In contrast, promoting fusion between the lysosome and plasma membrane dramatically increases infection. Our work demonstrates parallels between Plasmodium sporozoite entry of hepatocytes and infection by the excavate pathogen Trypanosoma cruzi and raises the question of whether convergent evolution has shaped host cell invasion by divergent pathogens.

Mycobacterium smegmatis PhoU Proteins Have Overlapping Functions in Phosphate Signaling and Are Essential.

Many bacteria regulate gene expression in response to phosphate availability using a two-component signal transduction system, the activity of which is controlled by interaction with the Pst phosphate specific transporter and a cytoplasmic protein PhoU. Mycobacterium tuberculosis, the causative agent of tuberculosis, requires its phosphate sensing signal transduction system for virulence and antibiotic tolerance, but the molecular mechanisms of phosphate sensing remain poorly characterized. M. smegmatis serves as a model for studying mycobacterial pathogens including M. tuberculosis. M. smegmatis encodes two proteins with similarity to PhoU, but it was unknown if both proteins participated in signal transduction with the phosphate-responsive SenX3-RegX3 two-component system. We constructed phoU single and double deletion mutants and tested expression of genes in the RegX3 regulon. Only the ΔphoU1ΔphoU2 mutant exhibited constitutive activation of all the RegX3-regulated genes examined, suggesting that M. smegmatis PhoU1 and PhoU2 have overlapping functions in inhibiting activity of the SenX3-RegX3 two-component system when phosphate is readily available. The ΔphoU1ΔphoU2 mutant also exhibited decreased tolerance to several anti-tubercular drugs. However, a complex plasmid swapping strategy was required to generate the ΔphoU1ΔphoU2 mutant, suggesting that either phoU1 or phoU2 is essential for in vitro growth of M. smegmatis. Using whole-genome sequencing, we demonstrated that all five of the ΔphoU1ΔphoU2 mutants we isolated had independent suppressor mutations predicted to disrupt the function of the Pst phosphate transporter, suggesting that in the absence of the PhoU proteins phosphate uptake by the Pst system is toxic. Collectively, our data demonstrate that the two M. smegmatis PhoU orthologs have overlapping functions in both controlling SenX3-RegX3 activity in response to phosphate availability and regulating phosphate transport by the Pst system. Our results suggest that M. smegmatis can serve as a tractable model for further characterization of the molecular mechanism of phosphate sensing in mycobacteria and to screen for compounds that would interfere with signal transduction and thereby increase the efficacy of existing anti-tubercular antibiotics.