To facilitate efficient allelic exchange of genetic information into a wild-type strain background, we improved upon and merged approaches using a temperature-sensitive plasmid and a counter-selectable marker in the chromosome. We first constructed intermediate strains of Escherichia coli K12 in which we replaced wild-type chromosomal sequences, at either the fimB-A or lacZ-A loci, with a newly constituted DNA cassette. The cassette consists of the sacB gene from Bacillus subtilis and the neomycin (kanamycin) resistance gene of Tn5, but, unlike another similar cassette, it lacks IS1 sequences. We found that sucrose sensitivity was highly dependent on incubation temperature and sodium chloride concentration. The DNA to be exchanged into the chromosome was first cloned into derivatives of plasmid pMAK705, a temperature-sensitive pSC101 replicon. The exchanges were carried out in two steps, first selecting for plasmid integration by standard techniques. In the second step, we grew the plasmid integrates under non-selective conditions at 42 degrees C, and then in the presence of sucrose at 30 degrees C, allowing positive selection for both plasmid excision and curing. Despite marked locus-specific strain differences in sucrose sensitivity and in the growth retardation due to the integrated plasmids, the protocol permitted highly efficient exchange of cloned DNA into either the fim or lac chromosomal loci. This procedure should allow the exchange of any DNA segment, in addition to the original or mutant allelic DNA, into any non-essential parts of the E. coli chromosome.

SummaryA survey of Haemophilus in¯uenzae strains indicated that around one-third of capsular strains and over two-thirds of non-typeable strains included sialic acid in their lipopolysaccharides (LPS). Mutation of the CMP-Neu5Ac synthetase gene (siaB ) resulted in a sialylation-de®cient phenotype. Isogenic pairs, wild type and siaB mutant of two non-typeable strains were used to demonstrate that sialic acid in¯uences resistance to the killing effect of normal human serum but has little effect on attachment to, or invasion of, cultured human epithelial cells or neutrophils. We determine for the ®rst time the site of attachment of sialic acid in the LPS of a non-typeable strain and report that a small proportion of glycoforms include two sialic acid residues in a disaccharide unit.

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.

We characterized the expression of a putative toxin of Bacillus anthracis, a member of the cholesteroldependent 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 ϳ30 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.

Serum-sensitive strains of Neisseria gonorrhoeae were incubated with suspensions of normal or chronic granulomatous disease human neutrophils in the absence or presence of fresh or heat-inactivated human serum; phagocytosis, gonococcal viability, and chemiluminescence were measured. Nonpiliated opaque or transparent gonococci (colony types 3 and 4, respectively) were used for phagocytic bactericidal assays. In the presence of 2.0% fresh human serum, normal neutrophils killed >90% of types 3 and 4 gonococci by 135 min. Serum alone at this concentration was not bactericidal. In the absence of serum, type 4 gonococci were not killed, whereas type 3 gonococci were killed to the same degree as in the presence of serum. Interestingly, heat-inactivated normal serum slightly inhibited phagocytic killing of type 3 gonococci. Results almost identical to those above were obtained when 5% fresh human serum deficient in complement component 7 was substituted for 2% normal autologous serum. This indicated that the later components of complement were not involved in the observed results. To investigate the mechanisms responsible for the intracellular killing of the gonococci, we used neutrophils from patients with chronic granulomatous disease. These neutrophils are deficient in an activable NADPH oxidase and do not produce bactericidal oxygen products upon phagocytic stimulation. Neutrophils from two unrelated boys with chronic granulomatous disease killed type 3 and 4 gonococci to the same degree as did normal neutrophils. As with normal neutrophils, serum was needed for killing type 4 organisms. As expected, neutrophils from these patients showed absolutely no increased chemiluminescence in the presence of type 3 or 4 gonococci, with or without serum. The effects of serum on gonococcus-induced chemiluminescence by normal neutrophils was also investigated. For these studies, in addition to type 3 and 4 gonococci, we also used transparent colony types of lightly (type 1) and heavily (type 2) piliated organisms. Chemiluminescence induced by type 1, 2, or 3 gonococci (i.e., gonococci possessing either pili or opacity-associated proteins, but not both) was augmented only slightly by serum and then only at low ratios of gonococci to neutrophils. On the other hand, chemiluminescence induced by type 4 gonococci (i.e., gonococci possessing neither pili nor opacity-associated proteins) was substantially increased in the presence of serum. Stimulation of chemiluminescence by type 1, 2, 3, or 4 gonococci was dose dependent in the absence or presence of serum. Heat-killed type 3 gonococci induced chemiluminescence to the same degree as did viable organisms. Since the gonococci used in this research was strongly catalase positive, as are gonococci in general, and since it was killed by chronic granulomatous disease neutrophils, the results indicate that gonococci can be effectively killed within neutrophils, i.e., within phagolysosomes, by nonoxidative bactericidal mechanisms. Whereas type 3 gonococci were phagocytized and killed by neutr...

We investigated the role of the protein II (P.11) family of gonococcal outer membrane proteins in the interaction of seven single P.11 variants of Neisseria gonorrhoeae FA1090 with human neutrophils in vitro. The abilities of nonpiliated gonococci to adhere to and be killed by neutrophils and to stimulate luminol-dependent chemiluminescence (CL) depended on the possession of at least one P.11. Gonococci lacking P.11 (i.e., P.II-) adhered poorly to and were not killed by neutrophils and induced only minimal CL. Although most P.11-containing (i.e., P.II+) variants adhered to, stimulated, and were readily killed by neutrophils, one variant, containing P.IIa, possessed none of these characteristics; it acted just like a P.IIvariant. No correlation was found between the colony opacity phenotype and the interaction of gonococci with neutrophils. Data from CL experiments suggest that the stimulatory effect of P.II was dominant over that of pili; i.e., piliated P.II+ gonococci were much more stimulatory than piliated P.IIgonococci. The results indicate that most but not all P.11 proteins mediate, in part or in full, the interaction of N. gonorrhoeae with human neutrophils, including adherence, stimulation of the neutrophil respiratory burst, and phagocytic killing.

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.Cholesterol-dependent cytolysins (CDCs) 4 are a family of pore-forming toxins from many organisms, including but not limited to the genera Archanobacterium, Bacillus, Clostridium, Listeria, and Streptococcus. Recently, work in vertebrates has revealed that CDCs and membrane attack complex/perforin superfamily domain-containing proteins share a similar fold, suggesting that vertebrates use a similar mechanism for defense against infection (1, 2). A common feature of the CDC family is the requirement of cholesterol in the membrane to form pores (3). In addition to cholesterol, certain members of the family also require a cellular receptor, such as CD59 for the toxin ILY from Streptococcus intermedius (4). The specific mechanism by which CDCs form pores is not completely resolved; however, what is generally known is that ring-shaped oligomerization at the cellular membrane is followed by large conformational changes in each unit of the oligomer, resulting in the insertion of a ␤-barrel into the cellular membrane (5). Pore formation results in a variety of downstream signaling effects, including but not limited to the influx of Ca 2ϩ into the cell (6).A good deal is known about structures of the prepore conformation of CDCs. The crystal structures of prepore PFO, from Clostridium perfringens, and ILY have previously been elucidated (7,8). Each structure shows a characteristic fourdomain architecture, in which domain 4 (D4) is involved in membrane recognition, domain 3 (D3) is involved in ␤...

SummaryNeisseria gonorrhoeae opacity-associated (Opa) proteins are a family of outer membrane proteins involved in gonococcal adherence to and invasion of human cells. We wanted to identify additional roles for Opa in the infectious process and used the yeast twohybrid system to identify human epithelial cell proteins that interact with Opa proteins. Although this system has been used successfully to identify many types of interacting proteins, it has not been used to screen a human cell cDNA library for binding partners of a prokaryotic outer membrane protein. Therefore, we were also interested in exploring the versatility of the yeast two-hybrid system in identifying bacteriahost interactions. Using OpaP from strain F62SF as bait, we screened a HeLa cell cDNA library for Opainteracting proteins (OIPs). We identified five different OIPs, designated OIP1-OIP5, two of which are homologous to human proteins -thyroid hormone receptor interacting protein (TRIP6) and pyruvate kinase isoenzyme M2 (PK). In the studies presented here, we investigated the interaction between Opa proteins and PK in more depth. Opa-PK interactions were confirmed by in vitro and in vivo assays independent of the yeast twohybrid system. Escherichia coli expressing six different Opa proteins from gonococcal strain FA1090 all bound more PK than Opa-negative E. coli in in vitro binding assays. Using anti-PK antibody and fluorescence microscopy, we showed that human epithelial cell PK co-localizes with intracellular Opaþ gonococci and E. coli expressing Opa proteins. Using a mutant of N. gonorrhoeae unable to grow on pyruvate or lactate, it appears that intracellular pyruvate is essential for gonococcal growth and survival. These results suggest a novel mechanism in bacterial pathogenesis, i.e. the requirement for direct molecular interaction with a host metabolic enzyme (PK) for the acquisition of an essential intracellular carbon source and growth substrate (pyruvate). These results demonstrate that the yeast two-hybrid system is a valuable tool for identifying biologically relevant interactions between bacteria and host proteins, providing valuable leads for further investigations into novel mechanisms of bacterial pathogenesis.