We isolated a rough variant of Mycobacterium abscessus CIP 104536T during experimental infection of mice. We show that this variant has lost the ability to produce glycopeptidolipids, is hyperlethal for C57BL/6 mice infected intravenously, and induces a strong tumor necrosis factor-alpha response by murine monocyte-derived macrophages.Mycobacterium abscessus (formerly Mycobacterium chelonae subsp. abscessus) is an emerging, rapidly growing mycobacterium that causes a wide spectrum of human infections, including skin and soft tissue infections (3, 26), lung infections (11, 24), and disseminated infections in patients either under immunosuppressive therapy (3, 23) or with a Mendelian syndrome conferring susceptibility to mycobacteria (5). Extended lung infections and disseminated infections raise serious therapeutic issues because M. abscessus strains are resistant to most antibiotics and are associated with a particular high fatality rate (3,23).M. abscessus organisms may be isolated with a smooth (S) or a rough (R) morphotype from clinical samples (23). Recently, Byrd and Lyons described an R strain of M. abscessus from an ileal granuloma in a patient with Crohn's disease, and they reported the spontaneous in vitro dissociation of this isolate into an S variant (4). Interestingly, these authors showed that the S variant was severely attenuated in its ability to infect the murine host and to persist in human monocytes. M. abscessus may thus undergo an S/R phase variation linked to pathogenicity. The molecular basis for the S or R appearance of M. abscessus, as well as the reason for the difference in pathogenicity of S and R variants, is presently unknown (12).We have recently used M. abscessus CIP 104536T (ϭATCC 19977T) (19) to study the immune control of M. abscessus infection in the murine host. This strain, which was provided by the Laboratoire de Référence des Mycobactéries (Institut Pasteur, Paris, France) has an S morphotype on ordinary solid media such as Trypticase soy agar (BioMérieux, Marcy l'Etoile, France) (Fig. 1A). Following the intravenous (i.v.) injection of 10 7 CFU of CIP 104536T into immunoglobulin chain knockout mice (15), we obtained mycobacterial colonies of the R morphotype from deep organs of several animals on day 90 following infection. We verified that these colonies were truly M. abscessus by partial hsp65 sequencing (21). One isolated colony (CIP 104536T-R) was selected, reisolated, and cryopreserved at Ϫ80°C using cryobeads (Mast Diagnostics, Reinfeld, Germany). The R morphotype of this variant was confirmed to be stable after 10 subcultures on solid media and three iterative in vivo passages. Thus, in contrast with previous studies reporting the switch of an R M. abscessus strain into an S morphotype (4), we show here that R variants may be selected in vivo from an M. abscessus strain with an S morphotype. Glycopeptidolipids (GPLs), also called C-mycosides or Jsubstances, represent up to 85% of the surface-exposed lipids in M. abscessus and a number of other nontuberculous mycobact...

We purified and characterized an extracellular hemolysin produced by Listeria monocytogenes. Hemolysin production was greatly enhanced by growing bacteria in resin (Chelex)-treated medium. This hemolysin was separated as a homogeneous protein of 60,000 daltons by using thiol-disulfide exchange affinity chromatography. This protein was a sulflydryl-activated toxin, termed listeriolysin 0, which shared the classical properties of other bacterial sulfhydryl-activated toxins: (i) inhibition by very low amounts of cholesterol; (ii) activation by reducing agents and suppression of the lytic activity by oxidation; (iii) antigenic cross-reactivity with streptolysin 0. However, listeriolysin 0 differed remarkably from the other sulfhydryl-activated toxins in that its cytolytic activity towards erythrocytes from various animal species was maximum at low pH (-5.5) and was undetectable at pH 7.0. This suggests that the lytic activity of the toxin in host tissues might be better expressed in the acidic microenvironment, including macrophage phagosomes where bacteria presumably replicate. Listeriolysin 0 was lethal to mice (50% lethal dose of ca. 0.8 ,ug) and induced a rapid inflammatory reaction when injected intradermally. These results favor the view that listeriolysin 0 might play a major role during intracellular replication of L. monocytogenes, ultimately promoting death of infected macrophages.

, a rapidly growing mycobacterium (RGM) and an opportunistic human pathogen, is responsible for a wide spectrum of clinical manifestations ranging from pulmonary to skin and soft tissue infections. This intracellular organism can resist the bactericidal defense mechanisms of amoebae and macrophages, an ability that has not been observed in other RGM. can up-regulate several virulence factors during transient infection of amoebae, thereby becoming more virulent in subsequent respiratory infections in mice. Here, we sought to identify the genes required for replication within amoebae. To this end, we constructed and screened a transposon () insertion library of an subspcies clinical isolate for attenuated clones. This approach identified five genes within the ESX-4 locus, which in encodes an ESX-4 type VII secretion system that exceptionally also includes the ESX conserved EccE component. To confirm the screening results and to get further insight into the contribution of ESX-4 to growth and survival in amoebae and macrophages, we generated a deletion mutant of that encodes a core structural element of ESX-4. This mutant was less efficient at blocking phagosomal acidification than its parental strain. Importantly, and in contrast to the wild-type strain, it also failed to damage phagosomes and showed reduced signs of phagosome-to-cytosol contact, as demonstrated by a combination of cellular and immunological assays. This study attributes an unexpected and genuine biological role to the underexplored mycobacterial ESX-4 system and its substrates.