Stochastic formation of Mycobacterium tuberculosis (Mtb) persisters achieves a high level of antibiotic-tolerance and serves as a source of multidrug-resistant (MDR) mutations. As conventional treatment is not effective against infections by persisters and MDR-Mtb, novel therapeutics are needed. Several approaches were proposed to kill persisters by altering their metabolism, obviating the need to target active processes. Here, we adapted a biofilm culture to model Mtb persister-like bacilli (PLB) and demonstrated that PLB underwent trehalose metabolism remodeling. PLB use trehalose as an internal carbon to biosynthesize central carbon metabolism intermediates instead of cell surface glycolipids, thus maintaining levels of ATP and antioxidants. Similar changes were identified in Mtb following antibiotic-treatment, and MDR-Mtb as mechanisms to circumvent antibiotic effects. This suggests that trehalose metabolism is associated not only with transient drug-tolerance but also permanent drug-resistance, and serves as a source of adjunctive therapeutic options, potentiating antibiotic efficacy by interfering with adaptive strategies.

The stringent response enables Mycobacterium tuberculosis (Mtb) to shut down its replication and metabolism under various stresses. Here we show that Mtb lacking the stringent response enzyme RelMtb was unable to slow its replication rate during nutrient starvation. Metabolomics analysis revealed that the nutrient-starved relMtb-deficient strain had increased metabolism similar to that of exponentially growing wild-type bacteria in nutrient-rich broth, consistent with an inability to enter quiescence. Deficiency of relMtb increased the susceptibility of mutant bacteria to killing by isoniazid during nutrient starvation and in the lungs of chronically infected mice. We screened a pharmaceutical library of over 2 million compounds for inhibitors of RelMtb and showed that the lead compound X9 was able to directly kill nutrient-starved M. tuberculosis and enhanced the killing activity of isoniazid. Inhibition of RelMtb is a promising approach to target M. tuberculosis persisters, with the potential to shorten the duration of TB treatment.

Carbapenems (imipenem, meropenem, biapenem, ertapenem, and doripenem) are β-lactam antimicrobial agents. Because carbapenems have the broadest spectra among all β-lactams and are primarily used to treat infections by multi-resistant Gram-negative bacteria, the emergence and spread of carbapenemases became a major public health concern. Carbapenemases are the most versatile family of β-lactamases that are able to hydrolyze carbapenems and many other β-lactams. According to the dependency of divalent cations for enzyme activation, carbapenemases can be divided into metallo-carbapenemases (zinc-dependent class B) and non-metallo-carbapenemases (zinc-independent classes A, C, and D). Many studies have provided various carbapenemase structures. Here we present a comprehensive and systematic review of three-dimensional structures of carbapenemase-carbapenem complexes as well as those of carbapenemases. We update recent studies in understanding the enzymatic mechanism of each class of carbapenemase, and summarize structural insights about regions and residues that are important in acquiring the carbapenemase activity.

BackgroundSensing and responding to environmental changes is a central aspect of cell division regulation. Mycobacterium tuberculosis contains eleven Ser/Thr kinases, two of which, PknA and PknB, are key signaling molecules that regulate cell division/morphology. One substrate of these kinases is Wag31, and we previously showed that partial depletion of Wag31 caused morphological changes indicative of cell wall defects, and that the phosphorylation state of Wag31 affected cell growth in mycobacteria. In the present study, we further characterized the role of the Wag31 phosphorylation in polar peptidoglycan biosynthesis.ResultsWe demonstrate that the differential growth among cells expressing different wag31 alleles (wild-type, phosphoablative, or phosphomimetic) is caused by, at least in part, dissimilar nascent peptidoglycan biosynthesis. The phosphorylation state of Wag31 is found to be important for protein-protein interactions between the Wag31 molecules, and thus, for its polar localization. Consistent with these results, cells expressing a phosphomimetic wag31 allele have a higher enzymatic activity in the peptidoglycan biosynthetic pathway.ConclusionsThe Wag31Mtb phosphorylation is a novel molecular mechanism by which Wag31Mtb regulates peptidoglycan synthesis and thus, optimal growth in mycobacteria.

Background Systemic cancer spread is preceded by the establishment of a permissive microenvironment in the target tissue of metastasis - the premetastatic niche. As crucial players in establishment of the pre-metastatic niche, myeloid derived suppressor cells (MDSC) release S100A8/A9, an exosomal protein that contributes to metastasis, angiogenesis, and immune suppression. We report the application of antibody-based single-photon emission computed tomography (SPECT) for detection of S100A8/A9 in vivo as an imaging marker for pre-metastatic tissue priming.Methods A syngeneic model system for invasive breast cancer with (4T1.2) or without (67NR) the tendency to form lung metastasis was established in BALB/c mice. A SPECT-probe has been generated and tested for visualization of S100A9 release. Tumor-associated changes in numbers and fuction of immune cells in pre-metastatic tissue were evaluated by flow cytometry and confocal microscopy.Results S100A8/A9 imaging reflected MDSC abundance and the establishment of an immunosuppressive environment in pre-metastatic lung tissue (activity 4T1.2 vs. healthy control: 0.95 vs. 0.45 %ID; p<0.001). The S100A8/A9 imaging signal in the pre-metastatic lung correlated with the subsequent metastatic tumor burden in the same organ (r2=0.788; p<0.0001). CCL2 blockade and the consecutive inhibition of premetastatic niche establishment was clearly depicted by S100A9-SPECT (lung activity untreated vs. treated: 2 vs, 1.4 %ID).Conclusion We report S100A8/A9 as a potent imaging biomarker for tumor-mediated immune remodeling with potential applications in basic research and clinical oncology.