RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in Mycobacterium smegmatis, and transcription start site (TSS) mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5’ untranslated region (UTR) and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the cis-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5’ end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5’ ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5’ UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression. The current paradigm for bacterial translation is based on an mRNA that includes an untranslated leader sequence containing the ribosome-binding site upstream of the initiation codon. We applied genome-scale approaches to map the protein-coding regions in the genomes of Mycobacterium smegmatis and Mycobacterium tuberculosis. We found that nearly one-quarter of mycobacterial transcripts are leaderless in mycobacterial species, thus indicating that ribosomes must recognize these mRNAs by a novel mechanism and suggesting that there are alternative modes of bacterial translation beyond the Escherichia coli paradigm. Our translational profiling showed that many mycobacterial proteins are mis-annotated, and also found many new genes encoding small proteins that had been previously overlooked, which are likely to play novel roles in diverse cellular processes. We also developed a new reporter system that provides mechanistic insights into translation initiation through deep sequencing. Our data show that leaderless translation is a robust process that is conserved in mycobacteria, that leaderless translation only requires that the mRNA begin with a start codon, and predict that mycobacteria encode hundreds of small proteins. This work will help us understand gene structure, genome organization and protein expression in bacteria, and how the translational machinery differs in different organisms.

The Mycobacterium tuberculosis Ser/Thr kinase PknB has been implicated in the regulation of cell growth and morphology in this organism. The extracytoplasmic domain of this membrane protein comprises four penicillin binding protein and Ser/Thr kinase associated (PASTA) domains, which are predicted to bind stem peptides of peptidoglycan. Using a comprehensive library of synthetic muropeptides, we demonstrate that the extracytoplasmic domain of PknB binds muropeptides in a manner dependent on the presence of specific amino acids at the second and third positions of the stem peptide, and on the presence of the sugar moiety N-acetylmuramic acid linked to the peptide. We further show that PknB localizes strongly to the mid-cell and also to the cell poles, and that the extracytoplasmic domain is required for PknB localization. In contrast to strong growth stimulation by conditioned medium, we observe no growth stimulation of M. tuberculosis by a synthetic muropeptide with high affinity for the PknB PASTAs. We do find a moderate effect of a high affinity peptide on resuscitation of dormant cells. While the PASTA domains of PknB may play a role in stimulating growth by binding exogenous peptidoglycan fragments, our data indicate that a major function of these domains is for proper PknB localization, likely through binding of peptidoglycan fragments produced locally at the mid-cell and the cell poles. These data suggest a model in which PknB is targeted to the sites of peptidoglycan turnover to regulate cell growth and cell division. Regulation of growth by Mycobacterium tuberculosis is important in the pathogenesis of tuberculosis (TB), including asymptomatic latent TB infection and active TB disease. The M. tuberculosis kinase PknB regulates cell growth and cell division by phosphorylating proteins involved in these processes to modify their function. The activity of PknB is thought to respond to extracellular stimuli by binding specific molecules with its extracytoplasmic domain. In this work we show that cell wall fragments bind to this domain, and that strong binding requires that these interacting molecules have specific molecular features. We demonstrate that a peptidoglycan fragment that binds strongly can stimulate growth of dormant bacteria, but that it does not affect growth of non-dormant bacteria. We also show that PknB localizes to the site of cell division and to the growing tip of the bacterium, where cell wall synthesis and degradation occur, and that the extracytoplasmic domain is required for this localization. These findings indicate that a major function of the extracytoplasmic domain of PknB is to place it at the sites of cell wall turnover, and suggest a model by which PknB can regulate growth and cell division, and thereby contribute to the pathogenesis of TB.

Background: Myrtus communis is a typical plant of Mediterranean area. The different parts of this plant such as berries, branches, and leaves have been used worldwide as a traditional/folk medicine for the treatment of various ailments and diseases. Methods: Ethanolic leaf extract of the plant was prepared by Soxhlet extraction method. Zone of inhibition, minimum inhibitory concentration and minimal bactericidal concentration were determined by well diffusion method and microplate alamar blue assay. GC-MS analysis was carried out to identify the compounds present in the extract. Microscopy and ImageJ software were used respectively for morphology and cell-length measurements. GraphPad Prism was used for statistical analysis. Results: The ethanolic extract showed strong inhibitory effect against Gram-positive and acid-fast bacteria with significant inhibition-zone size (9–25 mm), MIC (4.87–78 μg/ml), as well as MBC (0.3–20 mg/ml). However, no effect was observed on the growth of Gram-negative bacteria. The growth inhibition was found to be associated with the damage of cell wall as the extract-treated cells were sensitive to cell wall-targeting antibiotics and displayed the cell wall damage-depicting morphological defects. GC-MS analysis confirmed the presence of novel compounds in addition to the most representative compounds of the essential oils/extracts of M. communis of other country origins. Conclusion: These results demonstrate that M. communis leaf extract could be the source of compounds to be used for the treatment of Gram-positive bacterial infections. This is the first report, which provides insights into the mechanism of action of the extract in inhibiting the growth of Gram-positive bacteria.

Coronavirus disease 19 (COVID-19) is an ongoing global pandemic that is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The severity and mortality rates of COVID-19 are affected by several factors, such as respiratory diseases, diabetes, and hypertension. Bacterial coinfections are another factor that could contribute to the severity of COVID-19. Limited studies have investigated morbidity and mortality due to microbial coinfections in COVID-19 patients. Here, we retrospectively studied the effects of bacterial coinfections on intensive care unit (ICU)-admitted patients with COVID-19 in Asir province, Saudi Arabia. We analyzed electronic medical records of hospitalized patients with COVID-19 at Asir Central Hospital. A total of 34 patients were included, and the clinical data of 16 patients infected with SARS-CoV-2 only and 18 patients coinfected with SARS-CoV-2 and bacterial infections were analyzed in our study. Our data showed that the length of stay at the hospital for patients infected with both SARS-CoV-2 and bacterial infection was 35.2 days, compared to 16.2 days for patients infected with only SARS-CoV-2 (p = 0.0001). In addition, higher mortality rates were associated with patients in the coinfection group compared to the SARS-CoV-2-only infected group (50% vs. 18.7%, respectively). The study also showed that gram-negative bacteria are the most commonly isolated bacteria in COVID-19 patients. To conclude, this study found that individuals with COVID-19 who presented with bacterial infections are at higher risk for a longer stay at the hospital and potentially death. Further studies with a larger population are warranted to better understand the clinical outcomes of COVID-19 with bacterial infections.

Background. Apoptosis, a major form of programmed cell death, plays a vital role in regulating tissue development and maintenance of homeostasis in eukaryotes. Apoptosis can occur via a death receptor‐dependent extrinsic or a mitochondrial‐dependent intrinsic pathway and can be induced by various chemotherapeutic agents. In this study, the anticancer activity of Saussurea costus and its mode of intervention in human cancer cells of breast, colon, and liver were investigated. Results. In this study, the bioactives of S. costus leaves were extensively extracted in five solvents of different polarity. The cytotoxicity and anticancer effect of the extracted secondary metabolites were investigated against breast (MCF‐7), liver (HepG2), and colon (HCT116) cancer cell lines using a Sulphorhodamine B (SRB) assay. Secondary metabolites extracted using hexane, methanol, ethyl acetate, and chloroform had the highest cytotoxicity and thus the greatest anticancer effect on all the cancer cell lines tested (IC₅₀; ranging from 0.25 to 2.5 μg/ml), while butanol was comparatively less active (IC₅₀; ranging from 23.2 to 25.5 μg/ml). Further investigation using DNA flow cytometry and fluorescent microscopy revealed that the extract arrested the cells in the G₁ phase of cell cycle and induced apoptosis. Furthermore, the elevated expression level of proapoptotic proteins and decreased expression level of antiapoptotic proteins confirmed that the intrinsic (mitochondrial) pathway was involved in mediating the apoptosis of cancer cells upon treatment with S. costus extract. These results altogether suggest that S. costus could be a potential anticancer agent. Conclusion. These results suggest that the S. costus extract is the potential source of the secondary metabolites that could be used as anticancer agent to treat diverse cancers of breast, colon, and liver.

To persist and cause disease in the host, Mycobacterium tuberculosis must adapt to its environment during infection. Adaptations include changes in nutrient utilization and alterations in growth rate. M. tuberculosis Rv1422 is a conserved gene of unknown function that was found in a genetic screen to interact with the mce4 cholesterol uptake locus. The Rv1422 protein is phosphorylated by the M. tuberculosis Ser/Thr kinases PknA and PknB, which regulate cell growth and cell wall synthesis. Bacillus subtilis strains lacking the Rv1422 homologue yvcK grow poorly on several carbon sources, and yvcK is required for proper localization of peptidoglycan synthesis. Here we show that Mycobacterium smegmatis and M. tuberculosis strains lacking Rv1422 have growth defects in minimal medium containing limiting amounts of several different carbon sources. These strains also have morphological abnormalities, including shortened and bulging cells, suggesting a cell wall defect. In both mycobacterial species, the Rv1422 protein localizes uniquely to the growing cell pole, the site of peptidoglycan synthesis in mycobacteria. An M. tuberculosis ΔRv1422 strain is markedly attenuated for virulence in a mouse infection model, where it elicits decreased inflammation in the lungs and shows impaired bacterial persistence. These findings led us to name this gene cuvA ( c arbon u tilization and v irulence protein A ) and to suggest a model in which deletion of cuvA leads to changes in nutrient uptake and/or metabolism that affect cell wall structure, morphology, and virulence. Its role in virulence suggests that CuvA may be a useful target for novel inhibitors of M. tuberculosis during infection.