Autophagy is a cell biological pathway affecting immune responses. In vitro, autophagy acts as a cell-autonomous defense against Mycobacterium tuberculosis, but its role in vivo is unknown. Here we show that autophagy plays a dual role against tuberculosis: antibacterial and anti-inflammatory. M. tuberculosis infection of Atg5 fl/fl LysM-Cre + mice relative to autophagy-proficient littermates resulted in increased bacillary burden and excessive pulmonary inflammation characterized by neutrophil infiltration and IL-17 response with increased IL-1α levels. Macrophages from uninfected Atg5 fl/fl LysM-Cre + mice displayed a cell-autonomous IL-1α hypersecretion phenotype, whereas T cells showed propensity toward IL-17 polarization during nonspecific activation or upon restimulation with mycobacterial antigens. Thus, autophagy acts in vivo by suppressing both M. tuberculosis growth and damaging inflammation.utophagy is a fundamental cell biological process (1) with impact on aging, development, cancer, neurodegeneration, myodegeneration, metabolic disorders (2), idiopathic inflammatory diseases, and infection and immunity (3). Much of the physiological effects of autophagy are the result of degradative activities of autophagy (1), although biogenesis and secretory roles (4-6) of autophagy are beginning to be recognized (7). The execution of autophagy depends on factors collectively termed "Atg proteins," such as Atg5 (1) and Beclin 1 (Atg6) (8), whereas regulation of autophagy responds to various inputs via mammalian target of rapamycin (mTOR), including the presence of microbes (9), the TAB2/3-TAK1-IKK signaling axis (10), and processes downstream of pattern-recognition receptors and immune cytokine activation (3,(11)(12)(13).In the context of its immunological functions, autophagy acts in four principal ways (14). (i) Autophagy cooperates with conventional pattern-recognition receptors (PRRs), such as Toll-like receptors, RIG-I-like receptors (RLRs), and NOD-like receptors, and acts as both a regulator (11,12,15,16) and an effector of PRR signaling (17-19). (ii) Autophagy affects the presentation of cytosolic antigens in the context of MHC II molecules (20) in T-cell development, differentiation, polarization, and homeostasis (21,22). (iii) Most recently, autophagy has been shown to contribute to both the negative (6,7,(23)(24)(25) and positive (6, 7) regulation of unconventional secretion of the leaderless cytosolic proteins known as "alarmins," such as IL-1β and HMGB1. (iv) Autophagy can capture and eliminate intracellular microbes, including Mycobacterium tuberculosis (17, 26-29), which was one of the first two bacterial species (26, 30) to be recognized as targets for autophagic removal. This activity recently has been shown to depend on the recognition and capture of microbes by adaptors that represent a specialized subset of PRRs termed "sequestosome-like receptors" (SLRs) (31).M. tuberculosis is one of the first microbes recognized as being subject to elimination by immunological autophagy by murine and human...

Mycobacterium abscessus is an increasingly important cause of human disease; however, virulence determinants are largely uncharacterized. Previously, it was demonstrated that a rough, wild-type human clinical isolate (390R) causes persistent, invasive infection, while a smooth isogenic mutant (390S) has lost this capability. During serial passage of 390S, a spontaneous rough revertant was obtained, which was named 390V. This revertant regained the ability to cause persistent, invasive infection in human monocytes and the lungs of mice. Glycopeptidolipid (GPL), which plays a role in environmental colonization, was present in abundance in the cell wall of 390S, and was associated with sliding motility and biofilm formation. In contrast, a marked reduction in the amount of GPL in the cell wall of 390R and 390V was correlated with cord formation, a property associated with mycobacterial virulence. These results indicate that the ability to switch between smooth and rough morphologies may allow M. abscessus to transition between a colonizing phenotype and a more virulent, invasive form.

Bacillus anthracis, the agent of anthrax, produces a poly-Dglutamic acid capsule that has been implicated in virulence. Many strains missing pXO2 (96 kb), which harbors the capsule biosynthetic operon capBCAD, but carrying pXO1 (182 kb) that harbors the anthrax toxin genes, are attenuated in animal models. Also, noncapsulated strains are readily phagocytosed by macrophage cell lines, whereas capsulated strains are resistant to phagocytosis. We show that a strain carrying both virulence plasmids but deleted specifically for capBCAD is highly attenuated in a mouse model for inhalation anthrax. The parent strain and capsule mutant initiated germination in the lungs, but the capsule mutant did not disseminate to the spleen. A mutant harboring capBCAD but deleted for the cap regulators acpA and acpB was also significantly attenuated, in agreement with the capsule-negative phenotype during in vitro growth. Surprisingly, an acpB mutant, but not an acpA mutant, displayed an elevated LD 50 and reduced ability to disseminate, indicating that acpA and acpB are not true functional homologs and that acpB may play a larger role in virulence than originally suspected.

The ability to persist in the host after the establishment of infection is an important virulence determinant for mycobacteria.Mycobacterium abscessus is a rapidly growing mycobacterial species which causes a variety of clinical syndromes in humans. We have obtained a rough, wild-type human clinical isolate of M. abscessus (M. abscessus-R) and a smooth, attenuated mutant(M. abscessus-S) which spontaneously dissociated from the clinical isolate. We have found that M. abscessus-R is able to persist and multiply in a murine pulmonary infection model in contrast to M. abscessus-S, which is rapidly cleared. To understand the basis for this difference, we characterized the behavior of these variants in human tissue culture models of infection. M. abscessus-R is able to persist and multiply in human monocytes, while M. abscessus-S is deficient in this ability. Both of these variants are phagocytized by human monocytes. M. abscessus-R resides in a phagosome typical for pathogenic mycobacteria with a tightly adherent phagosomal membrane. In contrast,M. abscessus-S resides in a “loose” phagosome with the phagosomal membrane separated from the bacterial cell wall. BothM. abscessus variants also have distinctive growth patterns in a recently described fibroblast-mycobacterium microcolony assay, with M. abscessus-R exhibiting growth characteristics similar to those previously reported for virulent M. tuberculosis and M. abscessus-S exhibiting growth characteristics similar to those previously reported for avirulentM. tuberculosis. In both the monocyte infection assay and the murine pulmonary infection model, numerous infected mononuclear phagocyte aggregates develop at sites of M. abscessus-Rinfection, but are absent with M. abscessus-S infection. We conclude that a mutation has occurred in the M. abscessus-Svariant which has altered the ability of this organism to persist and multiply in host cells and that this may be related to the phenotypic changes we have observed in our tissue culture models of infection.

The inhalation of Francisella tularensis biovar A causes pneumonic tularemia associated with high morbidity and mortality rates in humans. Exposure to F. tularensis usually occurs by accident, but there is increasing awareness that F. tularensis may be deliberately released in an act of bioterrorism or war. The development of a vaccine against pneumonic tularemia has been limited by a lack of information regarding the mechanisms required to protect against this disease. Vaccine models for F. tularensis in inbred mice would facilitate investigations of the protective mechanisms and significantly enhance vaccine development. Intranasal vaccination with the attenuated live vaccine strain (LVS) of F. tularensis reproducibly protected BALB/c mice, but not C57BL/6 mice, against intranasal and subcutaneous challenges with a virulent clinical isolate of F. tularensis biovar A (NMFTA1). The resistance of LVS-vaccinated BALB/c mice to intranasal NMFTA1 challenge was increased 100-fold by boosting with live NMFTA1 but not with LVS. The protective response was specific for F. tularensis and required both CD4 and CD8 T cells. The vaccinated mice appeared outwardly healthy for more than 2 months after NMFTA1 challenge, even though NMFTA1 was recovered from more than half of the vaccinated mice. These results show that intranasal vaccination induces immunity that protects BALB/c mice from intranasal infection by F. tularensis biovar A.