Mycobacteria
Mycobacteria are a vast group of microorganisms characterized by a unique thick, hydrophobic cell wall rich in mycolic acids, which makes them highly resistant to environmental stresses. Even if most of them are innocuous environmental saprophytes, some of them, such as Mycobacterium leprae or Mycobacterium tuberculosis, have evolved to become formidable human pathogens with a very complex and still not well-characterized relationship with their host, while others, such as Mycobacterium avium, represent important emerging or opportunistic pathogens.
Guest-edited by Dr. Riccardo Manganelli, this collection of keynote research articles will highlight all aspects of mycobacterial biology, with particular focus on physiological aspects, such as stress response mechanisms, regulatory networks, and metabolic pathways, that might lead to a better understanding of the intriguing aspects of mycobacterial host-pathogen interaction and lead to the design of new strategies to fight these important pathogens.
Collection Contents
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A mycobacterial DivIVA domain-containing protein involved in cell length and septation
More LessMycobacterial cells elongate via polar deposition of cell wall material, similar to the filamentous Streptomyces species, which contain a tip-organizing centre. Coiled-coiled proteins such as DivIVA play an important role in this process. The genome of Mycobacterium tuberculosis , the causative agent of tuberculosis, encodes many coiled-coil proteins that are homologous to DivIVA with a potential role in mycobacterial cell elongation. Here we describe studies on Mycobacterium smegmatis MSMEG_2416, a homologue of M. tuberculosis Rv2927c. Two previous independent studies showed that MSMEG_2416 was involved in septation (subsequently referred to as sepIVA). Contrary to these previous reports, we found sepIVA to be dispensable for growth in laboratory media by generating a viable null mutant. The mutant strain did, however, show a number of differences, including a change in colony morphology and biofilm formation that could be reversed on complementation with sepIVA as well as Rv2927c, the sepIVA homologue from M. tuberculosis . However, analysis of cell wall lipids did not reveal any alterations in lipid profiles of the mutant strain. Microscopic examination of the mutant revealed longer cells with more septa, which occurred at irregular intervals, often generating mini-compartments, a profile similar to that observed in the previous studies following conditional depletion, highlighting a role for sepIVA in mycobacterial growth.
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MSMEG_2432 of Mycobacterium smegmatis mc2155 is a dual function enzyme that exhibits DD-carboxypeptidase and β-lactamase activities
Mycobacterial peptidoglycan (PG) is an unsolved puzzle due to its complex structure and involvement of multiple enzymes in the process of its remodelling. dd-Carboxypeptidases are low molecular mass penicillin-binding proteins (LMM-PBPs) that catalyzes the cleavage of terminal d-Ala of muramyl pentapeptide branches and thereby helps in the PG remodelling process. Here, we have assigned the function of a putative LMM-PBP, MSMEG_2432 of Mycobacterium smegmatis , by showing that it exhibits both dd-CPase and β-lactamase activities. Like conventional dd-CPase (PBP5 from E. coli), upon ectopic complementation in a deformed seven PBP deletion mutant of E. coli, MSMEG_2432 has manifested its ability to restore ~75 % of the cell population to their normal rod shape. Further, in vitro dd-CPase assay has confirmed its ability to release terminal d-Ala from the synthetic tripeptide and the peptidoglycan mimetic pentapeptide substrates ending with d-Ala-d-Ala. Also, elevated resistance against penicillins and cephalosporins upon ectopic expression of MSMEG_2432 suggests the presence of β-lactamase activity, which is further confirmed in vitro through nitrocefin hydrolysis assay. Moreover, it is found apparent that D169A substitution in MSMEG_2432 influences both of its in vivo and in vitro dd-CPase and β-lactamase activities. Thus, we infer that MSMEG_2432 is a dual function enzyme that possesses both dd-CPase and β-lactamase activities.
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Mycobacterium smegmatis moxifloxacin persister cells produce high levels of hydroxyl radical, generating genetic resisters selectable not only with moxifloxacin, but also with ethambutol and isoniazid
Bacterial antibiotic persister cells tolerate lethal concentrations of antibiotics but emerge as the antibiotic-sensitive population upon antibiotics withdrawal. However, the possibility of antibiotic-resistant genetic mutants emerging from the antibiotic persister population in the continued exposure to microbicidal concentrations of antibiotics needed investigation. We explored this possibility using the fast-growing Mycobacterium smegmatis as a model organism for Mycobacterium tuberculosis biology, as it is known to incur antibiotic-resistant mutations identical to and at identical target positions as found in the clinical isolates of M. tuberculosis . Here we report that the moxifloxacin (MXF) persister population generate significantly elevated levels of hydroxyl radical. Hydroxyl radical being a sequence-non-specific mutagen, resulted in the emergence of moxifloxacin-resistant genetic mutants at 8-log10 higher frequency from the persister population. Luria–Delbruck experiment (in modified format) confirmed that MXF-resistant mutants emerged de novo from the persister population and were not pre-existent. The nature of the mutations in the quinolone resistance determining region indicated that they were generated due to oxidative stress. These mutations were identical to and at identical positions as found in the clinical isolates of MXF-resistant M. tuberculosis . Interestingly, from the MXF persister population, resisters to microbicidal concentrations of ethambutol and isoniazid could also be selected. These observations implied that the significantly high levels of hydroxyl radical might have generated genome-wide mutations, creating a pool of mutants in the MXF persister population, facilitating selection of resisters to other antibiotics also. These findings may be of clinical relevance to the emergence of drug-resistant strains during prolonged tuberculosis treatment regimen with high doses of multiple antibiotics.
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A mycobacteriophage genomics approach to identify novel mycobacteriophage proteins with mycobactericidal properties
More LessMycobacteriophages that are specific to mycobacteria are sources of various effector proteins that are capable of eliciting bactericidal responses. We describe a genomics approach in combination with bioinformatics to identify mycobacteriophage proteins that are toxic to mycobacteria upon expression. A genomic library comprising phage genome collections was screened for clones capable of killing Mycobacterium smegmatis strain mc2155. We identified four unique clones: clones 45 and 12N (from the mycobacteriophage D29) and clones 66 and 85 (from the mycobacteriophage Che12). The gene products from clones 66 and 45 were identified as Gp49 of the Che12 phage and Gp34 of the D29 phage, respectively. The gene products of the other two clones, 85 and 12N, utilized novel open reading frames (ORFs) coding for synthetic proteins. These four clones (clones 45, 66, 85 and 12N) caused growth defects in M. smegmatis and Mycobacterium bovis upon expression. Clones with Gp49 and Gp34 also induced growth defects in Escherichia coli , indicating that they target conserved host machineries. Their expression induced various morphological changes, indicating that they affected DNA replication and cell division steps. We predicted that Gp34 is a Xis protein that is required in phage DNA excision from the bacterial chromosome. Gp49 is predicted to have an HTH motif with DNA-bending/twisting properties. We suggest that this methodology is useful to identify new phage proteins with the desired properties without laboriously characterizing the individual phages. It is universal and could be applied to other bacteria–phage systems. We speculate that the existence of a virtually unlimited number of phages with unique gene products could offer a cheaper and less hazardous alternative to explore new antimicrobial molecules.
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