Francisella tularensis, the causative agent of tularemia, survives and proliferates within macrophages of the infected host as part of its pathogenic strategy, through an intracellular life cycle that includes phagosomal escape and extensive proliferation within the macrophage cytosol. Various in vitro models of Francisellamacrophage interactions have been developed, using either opsonic or nonopsonic phagocytosis, and have generated discrepant results on the timing and extent of Francisella phagosomal escape. Here we have investigated whether either complement or antibody opsonization of the virulent prototypical type A strain Francisella tularensis subsp. tularensis Schu S4 affects its intracellular cycle within primary murine bone marrow-derived macrophages. Opsonization of Schu S4 with either human serum or purified IgG enhanced phagocytosis but restricted phagosomal escape and intracellular proliferation. Opsonization of Schu S4 with either fresh serum or purified antibodies redirected bacteria from the mannose receptor (MR) to the complement receptor CR3, the scavenger receptor A (SRA), and the Fc␥ receptor (Fc␥R), respectively. CR3-mediated uptake delayed maturation of the early Francisella-containing phagosome (FCP) and restricted phagosomal escape, while Fc␥R-dependent phagocytosis was associated with superoxide production in the early FCP and restricted phagosomal escape and intracellular growth in an NADPH oxidase-dependent manner. Taken together, these results demonstrate that opsonophagocytic receptors alter the intracellular fate of Francisella by delivering bacteria through phagocytic pathways that restrict phagosomal escape and intracellular proliferation.

Mycobacterium avium is an environmental organism and opportunistic pathogen with inherent resistance to drugs, environmental stresses, and the host immune response. To adapt to these disparate conditions, M. avium must control its transcriptional response to environmental cues. M. avium forms biofilms in various environmental settings, including drinking water pipes and potable water reservoirs. In this study, we investigated the role of the universal signaling molecule autoinducer-2 (AI-2) in biofilm formation by M. avium. The addition of the compound to planktonic M. avium cultures resulted in increased biofilm formation. Microarray and reverse transcriptase PCR studies revealed an upregulation of the oxidative stress response upon addition of AI-2. This suggests that the response to AI-2 might be related to oxidative stress, rather than quorum sensing. Consistent with this model, addition of hydrogen peroxide, a known stimulus of the oxidative stress response, to M. avium cultures resulted in elevated biofilm formation. These results suggest that AI-2 does not act as a quorum-sensing signal in M. avium. Instead, biofilm formation is triggered by environmental stresses of biotic and abiotic origins and AI-2 may exert effects on that level.Mycobacterium avium is ubiquitous in the environment, occurring in natural and urban water sources, as well as in soil, but also can act as an opportunistic pathogen (21, 37). It is related to the intracellular pathogens M. tuberculosis and M. leprae, the causative agents of tuberculosis and leprosy. The species M. avium is composed of several subspecies, including M. avium subsp. hominissuis, which causes disease in humans and other mammals, and M. avium subsp. paratuberculosis, a significant pathogen of livestock with an uncertain role in the etiology of human disease (53).Although M. avium mainly infects the immunocompromised, there have been increased reports of infections in people with no obvious predisposing factors (12). For example, hypersensitivity pneumonitis has been reported in otherwise healthy individuals. These infections occur due to exposure to aerosolized mycobacteria in indoor swimming pools, hot tubs, and metalworking fluids in industrial settings (1,29,45

The opportunistic pathogen Mycobacterium avium is a significant inhabitant of biofilms in drinking water distribution systems. M. avium expresses on its cell surface serovar-specific glycopeptidolipids (ssGPLs). Studies have implicated the core GPL in biofilm formation by M. avium and by other Mycobacterium species. In order to test this hypothesis in a directed fashion, three model systems were used to examine biofilm formation by mutants of M. avium with transposon insertions into pstAB (also known as nrp and mps). pstAB encodes the nonribosomal peptide synthetase that catalyzes the synthesis of the core GPL. The mutants did not adhere to polyvinyl chloride plates; however, they adhered well to plastic and glass chamber slide surfaces, albeit with different morphologies from the parent strain. In a model that quantified surface adherence under recirculating water, wild-type and pstAB mutant cells accumulated on stainless steel surfaces in equal numbers. Unexpectedly, pstAB mutant cells were >10-fold less abundant in the recirculating-water phase than parent strain cells. These observations show that GPLs are directly or indirectly required for colonization of some, but by no means all, surfaces. Under some conditions, GPLs may play an entirely different role by facilitating the survival or dispersal of nonadherent M. avium cells in circulating water. Such a function could contribute to waterborne M. avium infection.Members of the Mycobacterium avium complex (MAC), a group of closely related species and subspecies, are commonly isolated from water, food, soil, plants, and other samples. MAC species are associated with disease in birds and mammals, and some cause disease in susceptible humans. Sources of exposure include drinking water, spas, and soil. Mycobacteria are significant inhabitants of biofilms in these environments. They have been found in biofilm samples taken from water distribution systems, dental units, and medical devices at frequencies ranging from 69% to 95% of samples tested (1,11,12,25). Among the largest studies of mycobacteria in biofilms was that of Falkinham et al. (11). They sampled biofilms from posttreatment water pipes and customer water meters in eight U.S. cities. Mycobacteria were recovered from 69% of the samples and 100% of the sites. Slow-growing mycobacteria accounted for Ͼ90% of biofilm isolates. MAC species were the most common group recovered, accounting for 135 of the 267 (51%) individual Mycobacterium isolates from biofilm samples. In laboratory experiments, biofilm formation by MAC species has been correlated with chlorine resistance (24) and enhanced bronchial epithelial cell invasion (26). Despite its potential significance for human health, little is known about biofilm formation by the MAC.The cell walls of some Mycobacterium species have glycopeptidolipids (GPLs) that share a lipotetrapeptide core consisting of fatty acyl-NH-D-phenylalanine-D-allothreonine-D-alanine-L-alaninol (7). The alaninyl C terminus is rhamnosylated, and the allothreonine residue is linked to a...