Bacteria use a variety of secretion systems to transport proteins beyond their cell membrane to interact with their environment. For bacterial pathogens, these systems are key virulence determinants that transport bacterial proteins into host cells. Genetic screens to identify bacterial genes required for export have relied on enzymatic or fluorescent reporters fused to known substrates to monitor secretion. However, they cannot be used in analysis of all secretion systems, limiting the implementation across bacteria. Here, we introduce the first application of a modified form of whole colony MALDI-TOF MS to directly detect protein secretion from intact bacterial colonies. We show that this method is able to specifically monitor the ESX-1 system protein secretion system, a major virulence determinant in both mycobacterial and Gram-positive pathogens that is refractory to reporter analysis. We validate the use of this technology as a high throughput screening tool by identifying an ESAT-6 system 1-deficient mutant from a Mycobacterium marinum transposon insertion library. Furthermore, we also demonstrate detection of secreted proteins of the prevalent type III secretion system from the Gram-negative pathogen, Pseudomonas aeruginosa. This method will be broadly applicable to study other bacterial protein export systems and for the identification of compounds that inhibit bacterial protein secretion.

EsxA (ESAT-6) and EsxB (CFP-10) are virulence factors exported by the ESX-1 system in mycobacterial pathogens. In Mycobacterium marinum, an established model for ESX-1 secretion in Mycobacterium tuberculosis, genes required for ESX-1 export reside at the extended region of difference 1 (RD1) locus. In this study, a novel locus required for ESX-1 export in M. marinum was identified outside the RD1 locus. An M. marinum strain bearing a transposon-insertion between the MMAR_1663 and MMAR_1664 genes exhibited smooth-colony morphology, was deficient for ESX-1 export, was nonhemolytic, and was attenuated for virulence. Genetic complementation revealed a restoration of colony morphology and a partial restoration of virulence in cell culture models. Yet hemolysis and the export of ESX-1 substrates into the bacteriological medium in vitro as measured by both immunoblotting and quantitative proteomics were not restored. We show that genetic complementation of the transposon insertion strain partially restored the translocation of EsxA and EsxB to the mycobacterial cell surface. Our findings indicate that the export of EsxA and EsxB to the cell surface, rather than secretion into the bacteriological medium, correlates with virulence in M. marinum. Together, these findings not only expand the known genetic loci required for ESX-1 secretion in M. marinum but also provide an explanation for the observed disparity between in vitro ESX-1 export and virulence.T he ESAT-6 system-1 (ESX-1)/WXG-100 secretion system (Wss) is required for the virulence of both Gram-positive and mycobacterial pathogens (1-5). In mycobacterial pathogens the ESX-1 system likely promotes permeabilization of the phagosomal membrane allowing either the bacterial cell or bacterial products access to the cytosol of the macrophage (6-10). The ESX-1/Wss system is conserved and functional in nonpathogenic bacteria, where it promotes a range of activities, including conjugation (11-16).Protein substrates are translocated across the mycobacterial cytoplasmic membrane by the ESX-1 export system (3, 5). Several genes are required for protein translocation, disruption of which results in loss of substrate secretion into the bacteriological medium (culture supernatant) in vitro (3)(4)(5)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). It is unknown how the protein substrates are secreted across the mycolate outer membrane (MOM) out of the mycobacterial cell or if the known ESX genes promote MOM translocation. Likewise, it is not clear if the ESX-1 substrates are true exoproteins (released from the cell) or extrinsically associated with the MOM and shed into the bacteriological medium (27-29). Indeed, in addition to the culture supernatant, ESX-1 substrates have been localized to the mycobacterial cell wall and associated with the surface of the mycobacterial cell (28-31). It is unclear which population of ESX-1 substrates mediates virulence. The active secretion of ESX-1 substrates into the phagosome or cytosol of the macrophage has not been routinely observed.In the human ...

bMycobacterium marinum is a waterborne mycobacterial pathogen. Due to their common niche, protozoa likely represent natural hosts for M. marinum. We demonstrate that the ESX-1 secretion system is required for M. marinum pathogenesis and that M. marinum utilizes actin-based motility in amoebae. Therefore, at least two virulence pathways used by M. marinum in macrophages are conserved during M. marinum infection of amoebae. Mycobacterial pathogens are responsible for some of the leading causes of death by infectious disease. The majority of these deaths are caused by mycobacterial species within the Mycobacterium tuberculosis complex (MTC) (3). However, mycobacterial species in the environment, including atypical or nontuberculous mycobacteria (NTM), pose an emerging disease threat (19). Little is understood about the basic molecular virulence mechanisms employed by environmental mycobacterial pathogens.Mycobacterium marinum is a waterborne pathogen that causes a tuberculosis (TB)-like infection in ectotherms and is an occasional opportunistic human pathogen (21). M. marinum is related to M. tuberculosis and is used to model aspects of MTC pathogenesis (4,7,21,23,27). Free-living amoebae (FLA), including Acanthamoeba castellanii, are professional phagocytes (12). Pathogenic bacteria, including M. marinum and other NTM, have been recovered from samples of water colonized by free-living amoebae (8,26). Several mycobacterial species are established amoebaresistant bacteria (ARB) and resist destruction by FLA (1,6,12,17,18). It has been posited that protozoa serve as a reservoir for mycobacteria in the environment (17).It is probable that M. marinum naturally interacts with protozoa, including A. castellanii, that share an environmental niche. It was demonstrated that M. marinum are pathogenic to A. castellanii (6, 17, 20, 28). The molecular mechanisms underlying this interaction have not been well established.We hypothesized that virulence mechanisms required for infection of macrophages by M. marinum would also be required for pathogenesis of A. castellanii. The ESX-1 protein secretion system is required by mycobacteria and other Gram-positive pathogens to cause disease (2,11,13,16,24). Amoebae were infected (multiplicity of infection [MOI] of 1) with either the wild-type (WT) strain or an attenuated RD1 deletion (⌬RD1) strain of M. marinum, which bears a deletion in components and substrates of the ESX-1 system ( Fig. 1A) (27). M. marinum replicated roughly three logs in A. castellanii over the 72-h experiment, with an average generation time of 7 h. The ⌬RD1 strain replicated approximately two logs in A. castellanii over the 72-h experiment, with an average generation time of 12 h. Following an early rapid growth phase for both strains, the bacteria were maintained at relatively constant levels throughout the experiment. However, the ⌬RD1 strain failed to reach the levels of growth achieved by the wild-type strain (Fig. 1A). Significant differences in growth between the WT and ⌬RD1 strains were observed at 48 (P Ͻ...

The Williamsburg, Virginia, wastewater treatment plant (WWTP) has periodically experienced erratic disinfection and persistence of fecal coliform bacteria in the presence of apparently adequate levels of disinfectant in the effluent. Several reasons for disinfection problems were previously investigated. This paper describes the results of a study of two factors that could affect disinfection in this plant: (1) the potential for the development of disinfection-resistant fecal coliforms in the operation of this specific WWTP and (2) the nature of the wastewater matrix that this particular WWTP handles that could interfere with chlorine disinfection. The study suggested that the WWTP oxidation towers supported growth or recovery of fecal coliform bacteria. This bacterial subpopulation seemed to have increased resistance to routine chlorine disinfection. Inactivation rate constants calculated for periods between 15 and 45 minutes after chlorine addition were significantly lower for fecal coliforms originating from oxidation towers than for fecal coliforms from other sources. This is, to our knowledge, the first report of such conditions created by a treatment process. The study of the plant-specific matrix determined no statistically significant effects on disinfection. Water Environ. Res., 71, 277 (1999).