Mab_3083c Is a Homologue of RNase J and Plays a Role in Colony Morphotype, Aggregation, and Sliding Motility of Mycobacterium abscessus.

Mycobacterium abscessus is an opportunistic pathogen causing human diseases, especially in immunocompromised patients. M. abscessus strains with a rough morphotype are more virulent than those with a smooth morphotype. Morphotype switch may occur during a clinical infection. To investigate the genes involved in colony morphotype switching, we performed transposon mutagenesis in a rough clinical strain of M. abscessus. A morphotype switching mutant (smooth) named mab_3083c::Tn was obtained. This mutant was found to have a lower aggregative ability and a higher sliding motility than the wild type strain. However, its glycopeptidolipid (GPL) content remained the same as those of the wild type. Complementation of the mutant with a functional mab_3083c gene reverted its morphotype back to rough, indicating that mab_3083c is associated with colony morphology of M. abscessus. Bioinformatic analyses showed that mab_3083c has a 75.4% identity in amino acid sequence with the well-characterized ribonuclease J (RNase J) of M. smegmatis (RNase JMsmeg). Complementation of the mutant with the RNase J gene of M. smegmatis also switched its colony morphology from smooth back to rough. These results suggest that Mab_3083c is a homologue of RNase J and involved in regulating M. abscessus colony morphotype switching.

Cross-talk between bacterial two-component systems drives stepwise regulation of flagellar biosynthesis in swarming development.

Swarming motility is a mode of bacterial movement over a solid surface driven by rotating flagella in a coordinated manner. Bacteria can use two-component system (TCS), which typically comprises a sensor kinase and a specific cognate response regulator, to properly react to environmental changes. We previously showed that the TCS RssAB suppresses flagellar biosynthesis master regulator flhDC specifically in swarming lag phase to control surface migration timing without affecting expansion rate in Serratia marcescens swarming development. Here we demonstrate that the TCS QseBC, which has been found in several human pathogens involved in flagellar and virulence regulation, has cross-talk with RssAB. We demonstrate that the phosphorylated QseB repressed flhDC expression, reducing swarming migration rate with modest effect on migration initiation. Unexpectedly, the QseC can dephosphorylate non-cognate response regulator RssB. Deletion of qseC prolonged RssAB signaling, reduced flhDC expression, and delayed migration initiation. Our data suggest that QseC is a flagellar biosynthesis activator by de-repressing RssB âˆ¼ P and QseB âˆ¼ P respectively in lag and migration phases in a stage-specific manner in swarming development.

Subinhibitory Doses of Aminoglycoside Antibiotics Induce Changes in the Phenotype of Mycobacterium abscessus.

Subinhibitory doses of antibiotics have been shown to cause changes in bacterial morphology, adherence ability, and resistance to antibiotics. In this study, the effects of subinhibitory doses of aminoglycoside antibiotics on Mycobacterium abscessus were investigated. The treatment of M. abscessus cells with subinhibitory doses of amikacin was found to change their colony from a smooth to a rough morphotype and increase their ability to adhere to a polyvinylchloride plate, aggregate in culture, and resist phagocytosis and killing by macrophages. M. abscessus cells treated with a subinhibitory dose of amikacin also became more potent in Toll-like receptor 2 (TLR-2) stimulation, leading to increased tumor necrosis factor alpha (TNF-α) production by macrophages. The MAB_3508c gene was shown to play a role in mediating these phenotypic changes, as its expression in M. abscessus cells was increased when they were treated with a subinhibitory dose of amikacin. In addition, overexpression of MAB_3508c in M. abscessus cells caused changes similar to those induced by subinhibitory doses of amikacin, including a switch from smooth to rough colony morphology, increased ability to aggregate in liquid culture, decreased motility, and increased resistance to killing by macrophages. These findings suggest the importance of using sufficient doses of antibiotics for the treatment of M. abscessus infections.

Ganoderma lucidum stimulates NK cell cytotoxicity by inducing NKG2D/NCR activation and secretion of perforin and granulysin.

Ganoderma lucidum (G. lucidum) is a medicinal mushroom long used in Asia as a folk remedy to promote health and longevity. Recent studies indicate that G. lucidum activates NK cells, but the molecular mechanism underlying this effect has not been studied so far. To address this question, we prepared a water extract of G. lucidum and examined its effect on NK cells. We observed that G. lucidum treatment increases NK cell cytotoxicity by stimulating secretion of perforin and granulysin. The mechanism of activation involves an increased expression of NKG2D and natural cytotoxicity receptors (NCRs), as well as increased phosphorylation of intracellular MAPKs. Our results indicate that G. lucidum induces NK cell cytotoxicity against various cancer cell lines by activating NKG2D/NCR receptors and MAPK signaling pathways, which together culminate in exocytosis of perforin and granulysin. These observations provide a cellular and molecular mechanism to account for the reported anticancer effects of G. lucidum extracts in humans.

Mab_3168c, a putative acetyltransferase, enhances adherence, intracellular survival and antimicrobial resistance of Mycobacterium abscessus.

Mycobacterium abscessus is a non-tuberculous mycobacterium. It can cause diseases in both immunosuppressed and immunocompetent patients and is highly resistant to multiple antimicrobial agents. M. abscessus displays two different colony morphology types: smooth and rough morphotypes. Cells with a rough morphotype are more virulent. The purpose of this study was to identify genes responsible for M. abscessus morphotype switching. With transposon mutagenesis, a mutant with a Tn5 inserted into the promoter region of the mab_3168c gene was found to switch its colonies from a rough to a smooth morphotype. This mutant had a higher sliding motility but a lower ability to form biofilms, aggregate in culture, and survive inside macrophages. Results of bioinformatic analyses suggest that the putative Mab_3168c protein is a member of the GCN5-related N-acetyltransferase superfamily. This prediction was supported by the demonstration that the mab_3168c gene conferred M. abscessus and M. smegmatis cells resistance to amikacin. The multiple roles of mab_3168c suggest that it could be a potential target for development of therapeutic regimens to treat diseases caused by M. abscessus.

Activation of an NLRP3 inflammasome restricts Mycobacterium kansasii infection.

Mycobacterium kansasii has emerged as an important nontuberculous mycobacterium pathogen, whose incidence and prevalence have been increasing in the last decade. M. kansasii can cause pulmonary tuberculosis clinically and radiographically indistinguishable from that caused by Mycobacterium tuberculosis infection. Unlike the widely-studied M. tuberculosis, little is known about the innate immune response against M. kansasii infection. Although inflammasome activation plays an important role in host defense against bacterial infection, its role against atypical mycobacteria remains poorly understood. In this report, the role of inflammasome activity in THP-1 macrophages against M. kansasii infection was studied. Results indicated that viable, but not heat-killed, M. kansasii induced caspase-1-dependent IL-1ß secretion in macrophages. The underlying mechanism was found to be through activation of an inflammasome containing the NLR (Nod-like receptor) family member NLRP3 and the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). Further, potassium efflux, lysosomal acidification, ROS production and cathepsin B release played a role in M. kansasii-induced inflammasome activation. Finally, the secreted IL-1ß derived from caspase-1 activation was shown to restrict intracellular M. kansasii. These findings demonstrate a biological role for the NLRP3 inflammasome in host defense against M. kansasii.