Control of pili and sialyltransferase expression in Neisseria gonorrhoeae is mediated by the transcriptional regulator CrgA.

Contact-regulated gene A (CrgA) is a transcriptional regulator present in the pathogenic Neisseria that functions as both an activator and a repressor of transcription following contact with host cells. While its mechanism of action has been studied extensively in Neisseria meningitidis, the specific subset of genes that CrgA targets has been debated. Although the majority of these constitute virulence genes, suggesting that CrgA is important in pathogenesis, no study to date has examined the effects of CrgA in Neisseria gonorrhoeae. In this report, we generated a knockout mutant of crgA (ΔcrgA) in the serum-sensitive gonococcal strain F62. crgA deletion resulted in a reduction in the transcript and protein levels of the primary pilin component pilE via mechanisms that were both contact-dependent and -independent. In contrast, ΔcrgA overexpressed the main determinant of serum resistance in F62, lipooligosaccharide sialyltransferase (Lst). CrgA-mediated lst repression was direct as both recombinant and native CrgA bound to the lst promoter at multiple locations in EMSA and ChIP assays respectively. The increase in Lst levels associated with crgA deletion correlated with enhanced protection against killing by normal human serum. These data suggest a role for CrgA in virulence regulation during both cell adherence and planktonic growth.

Rapid sporulation of Bacillus anthracis in a high iron, glucose-free medium.

Spores are the infectious form of Bacillus anthracis (BA), causing cutaneous, inhalation and gastrointestinal anthrax. Because of the possible use of BA spores in a bioterrorism attack, there is considerable interest in studying spore biology. In the laboratory, however, it takes a number of days to prepare spores. Standard sporulation protocols, such as the use of 'PA broth', allow sporulation of BA to occur in 3 to 5 days. Another method employs growth of BA on plates in the dark for several days until they have efficiently sporulated. In efforts to determine the effect of iron on gene expression in BA, we grew BA Sterne strain 7702 in a minimal defined medium (CDM; Koppisch et al., 2005) with various concentrations of iron and glucose. As part of our initial observations, we monitored BA sporulation in CDM via light microscopy. In glucose-free CDM containing 1.5mM Fe(NO(3))(3) (CDM-Fe), >95% of the BA sporulated by 30 h; a far shorter time period than expected. We pursued this observation and we further characterized spores derived from PA and CDM-Fe media. Purified spores derived from PA or CDM-Fe had similar morphologies when viewed by light or electron microscopy, and were equally resistant to harsh conditions including heat (65 degrees C), ice and fresh 30% H(2)O(2). Spore viability in long term cold storage in water was similar for the two spore preparations. Extracted spore coat proteins were evaluated by SDS-PAGE and silver staining, which revealed distinct protein profiles for PA and CDM-Fe spore coat extracts. ELISA assays were done to compare the interaction of the two spore preparations with rabbit antiserum raised against UV-killed Sterne strain 7702 spores prepared in PA medium. Spores from both media reacted identically with this antiserum. Finally, the interaction and fate of spores incubated with macrophages in vitro was very similar. In summary, BA spores induced in CDM-Fe or in PA medium are similar by several criteria, but show distinct extractable coat proteins. CDM-Fe liquid medium can be used for rapid production of BA spores, and could save considerable time in spore research studies.

Human alpha-defensins inhibit hemolysis mediated by cholesterol-dependent cytolysins.

Many pathogenic gram-positive bacteria release exotoxins that belong to the family of cholesterol-dependent cytolysins. Here, we report that human alpha-defensins HNP-1 to HNP-3 acted in a concentration-dependent manner to protect human red blood cells from the lytic effects of three of these exotoxins: anthrolysin O (ALO), listeriolysin O, and pneumolysin. HD-5 was very effective against listeriolysin O but less effective against the other toxins. Human alpha-defensins HNP-4 and HD-6 and human beta-defensin-1, -2, and -3 lacked protective ability. HNP-1 required intact disulfide bonds to prevent toxin-mediated hemolysis. A fully linearized analog, in which all six cysteines were replaced by aminobutyric acid (Abu) residues, showed greatly reduced binding and protection. A partially unfolded HNP-1 analog, in which only cysteines 9 and 29 were replaced by Abu residues, showed intact ALO binding but was 10-fold less potent in preventing hemolysis. Surface plasmon resonance assays revealed that HNP-1 to HNP-3 bound all three toxins at multiple sites and also that solution-phase HNP molecules could bind immobilized HNP molecules. Defensin concentrations that inhibited hemolysis by ALO and listeriolysin did not prevent these toxins from binding either to red blood cells or to cholesterol. Others have shown that HNP-1 to HNP-3 inhibit lethal toxin of Bacillus anthracis, toxin B of Clostridium difficile, diphtheria toxin, and exotoxin A of Pseudomonas aeruginosa; however, this is the first time these defensins have been shown to inhibit pore-forming toxins. An "ABCDE mechanism" that can account for the ability of HNP-1 to HNP-3 to inhibit so many different exotoxins is proposed.

Cellular functions and X-ray structure of anthrolysin O, a cholesterol-dependent cytolysin secreted by Bacillus anthracis.

Anthrolysin O (ALO) is a pore-forming, cholesterol-dependent cytolysin (CDC) secreted by Bacillus anthracis, the etiologic agent for anthrax. Growing evidence suggests the involvement of ALO in anthrax pathogenesis. Here, we show that the apical application of ALO decreases the barrier function of human polarized epithelial cells as well as increases intracellular calcium and the internalization of the tight junction protein occludin. Using pharmacological agents, we also found that barrier function disruption requires increased intracellular calcium and protein degradation. We also report a crystal structure of the soluble state of ALO. Based on our analytical ultracentrifugation and light scattering studies, ALO exists as a monomer. Our ALO structure provides the molecular basis as to how ALO is locked in a monomeric state, in contrast to other CDCs that undergo antiparallel dimerization or higher order oligomerization in solution. ALO has four domains and is globally similar to perfringolysin O (PFO) and intermedilysin (ILY), yet the highly conserved undecapeptide region in domain 4 (D4) adopts a completely different conformation in all three CDCs. Consistent with the differences within D4 and at the D2-D4 interface, we found that ALO D4 plays a key role in affecting the barrier function of C2BBE cells, whereas PFO domain 4 cannot substitute for this role. Novel structural elements and unique cellular functions of ALO revealed by our studies provide new insight into the molecular basis for the diverse nature of the CDC family.

The Bacillus anthracis cholesterol-dependent cytolysin, Anthrolysin O, kills human neutrophils, monocytes and macrophages.

BACKGROUND: Bacillus anthracis is an animal and human pathogen whose virulence is characterized by lethal and edema toxin, as well as a poly-glutamic acid capsule. In addition to these well characterized toxins, B. anthracis secretes several proteases and phospholipases, and a newly described toxin of the cholesterol-dependent cytolysin (CDC) family, Anthrolysin O (ALO). RESULTS: In the present studies we show that recombinant ALO (rALO) or native ALO, secreted by viable B. anthracis, is lethal to human primary polymorphonuclear leukocytes (PMNs), monocytes, monocyte-derived macrophages (MDMs), lymphocytes, THP-1 monocytic human cell line and ME-180, Detroit 562, and A549 epithelial cells by trypan blue exclusion or lactate dehydrogenase (LDH) release viability assays. ALO cytotoxicity is dose and time dependent and susceptibility to ALO-mediated lysis differs between cell types. In addition, the viability of monocytes and hMDMs was assayed in the presence of vegetative Sterne strains 7702 (ALO+), UT231 (ALO-), and a complemented strain expressing ALO, UT231 (pUTE544), and was dependent upon the expression of ALO. Cytotoxicity of rALO is seen as low as 0.070 nM in the absence of serum. All direct cytotoxic activity is inhibited by the addition of cholesterol or serum concentration as low as 10%. CONCLUSION: The lethality of rALO and native ALO on human monocytes, neutrophils, macrophages and lymphocytes supports the idea that ALO may represent a previously unidentified virulence factor of B. anthracis. The study of other factors produced by B. anthracis, along with the major anthrax toxins, will lead to a better understanding of this bacterium's pathogenesis, as well as provide information for the development of antitoxin vaccines for treating and preventing anthrax.

Anthrolysin O and other gram-positive cytolysins are toll-like receptor 4 agonists.

Exposure of bone marrow-derived macrophages (BMDMs) to low concentrations of Bacillus anthracis lethal toxin (LT), whose catalytic subunit is lethal factor (LF), results in induction of a robust apoptotic response dependent on activation of Toll-like receptor (TLR)4. A similar TLR4-dependent apoptotic response is observed when BMDMs are infected with live B. anthracis (Sterne strain). However, TLR4 is considered to be a specific signaling receptor for lipopolysaccharide (LPS), a typical product of gram-negative bacteria, whereas B. anthracis is gram-positive. To understand how B. anthracis can activate TLR4, we analyzed its culture supernatants and found them to contain a potent TLR4-stimulating activity that can also induce apoptosis in macrophages in which the antiapoptotic p38 MAP kinase (whose activation is prevented by LF) was inhibited. Purification of this activity suggested it consists of anthrolysin O (ALO), a member of the cholesterol-dependent cytolysin (CDC) family. We show that recombinant ALO can activate TLR4 in a manner independent of LPS contamination and, together with LT, can induce macrophage apoptosis. We also provide genetic evidence that ALO is required for induction of macrophage apoptosis in response to infection with live B. anthracis and that other CDC family members share the ability to activate TLR4.

Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin.

We characterized the expression of a putative toxin of Bacillus anthracis, a member of the cholesterol-dependent cytolysin (CDC) family, which includes listeriolysin O, perfringolysin O, and streptolysin O. We named this cytotoxin anthrolysin O (ALO). Although B. anthracis expresses minimal hemolytic activity in clinical settings, we show that Sterne strain 7702 expresses hemolytic activity when grown in brain heart infusion broth or in other rich bacteriologic media, but it secretes barely detectable amounts of hemolysin when grown in Luria-Bertani (LB) broth. Glucose supplementation of LB broth increases the amount of secreted hemolytic activity. Expression of hemolytic activity is maximal during mid- to late-log phase and decreases in the stationary phase. These observations are supported, in part, by semiquantitative reverse transcriptase PCR of alo mRNA. Hemolytic activity in growth supernatants was increased in the presence of reducing agent and almost totally inhibited in a dose-dependent manner by cholesterol; both of these activities are characteristic of a CDC toxin. A mutant of Sterne strain 7702, strain UT231, in which the alo gene was deleted and replaced by a kanamycin cassette, secreted barely detectable hemolytic activity into the growth medium. When strain UT231 was complemented in trans with native alo on a low-copy-number plasmid [strain UT231(pUTE554)], it regained the ability to secrete hemolytic activity, indicating that ALO is the major hemolysin secreted by this strain of B. anthracis in rich media in vitro. To further support the alo gene product being a hemolysin, recombinant B. anthracis ALO (rALO) purified from Escherichia coli was extremely active against washed human erythrocytes, with complete hemolysis detected at approximately 30 molecules of rALO per erythrocyte. Considering the virulence roles of CDCs for other gram-positive bacteria, we speculate that ALO may have a role in anthrax virulence.