dWhooping cough results from infection of the respiratory tract with Bordetella pertussis, and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrations in vitro, ACT has not been observed or quantified in vivo, and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human and in vitro data, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ϳ10 8 CFU/ml B. pertussis and 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ϳ10 8 CFU/ml of a laboratory strain of B. pertussis was cultured in vitro, ACT production was detected in 60 min and reached a plateau of ϳ60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis.

The extracytoplasmic assembly of the Dot/Icm type IVb secretion system (T4SS) of Legionella pneumophila is dependent on correct disulfide bond (DSB) formation catalyzed by a novel and essential disulfide bond oxidoreductase DsbA2 and not by DsbA1, a second nonessential DSB oxidoreductase. DsbA2, which is widely distributed in the microbial world, is phylogenetically distinct from the canonical DsbA oxidase and the DsbC protein disulfide isomerase (PDI)/reductase of Escherichia coli. Here we show that the extended N-terminal amino acid sequence of DsbA2 (relative to DsbA proteins) contains a highly conserved 27-amino-acid dimerization domain enabling the protein to form a homodimer. Complementation tests with E. coli mutants established that L. pneumophila dsbA1, but not the dsbA2 strain, restored motility to a dsbA mutant. In a protein-folding PDI detector assay, the dsbA2 strain, but not the dsbA1 strain, complemented a dsbC mutant of E. coli. Deletion of the dimerization domain sequences from DsbA2 produced the monomer (DsbA2N), which no longer exhibited PDI activity but complemented the E. coli dsbA mutant. PDI activity was demonstrated in vitro for DsbA2 but not DsbA1 in a nitrocefin-based mutant TEM β-lactamase folding assay. In an insulin reduction assay, DsbA2N activity was intermediate between those of DsbA2 and DsbA1. In L. pneumophila, DsbA2 was maintained as a mixture of thiol and disulfide forms, while in E. coli, DsbA2 was present as the reduced thiol. Our studies suggest that DsbA2 is a naturally occurring bifunctional disulfide bond oxidoreductase that may be uniquely suited to the majority of intracellular bacterial pathogens expressing T4SSs as well as in many slow-growing soil and aquatic bacteria.

We investigated whether nematodes contribute to the persistence, differentiation and amplification of Legionella species in soil, an emerging source for Legionnaires’ disease. Here we show that Legionella spp. colonize the intestinal tracts of Caenorhabditis nematodes leading to worm death. Susceptibility to Legionella is influenced by innate immune responses governed by the p38 mitogen-activated protein kinase and insulin/insulin growth factor-1 receptor signaling pathways. We also show that L. pneumophila colonizes the intestinal tract of nematodes cultivated in soil. To distinguish between transient infection and persistence, plate-fed and soil-extracted nematodes fed fluorescent strains of L. pneumophila were analyzed. Bacteria replicated within the nematode intestinal tract, did not invade surrounding tissue, and were excreted as differentiated forms that were transmitted to offspring. Interestingly, the ultrastructural features of the differentiated bacterial forms were similar to cyst-like forms observed within protozoa, amoeba and mammalian cell lines. While intestinal colonization of L. pneumophila dotA and icmT mutant strains did not alter the survival rate of nematodes in comparison to wild-type strains, nematodes colonized with the dot/icm mutant strains exhibited significantly increased levels of germline apoptosis. Taken together, these studies show that nematodes may serve as natural hosts for these organisms and thereby contribute to their dissemination in the environment and suggest that the remarkable ability of L. pneumophila to subvert host cell signaling and evade mammalian immune responses evolved through the natural selection associated with cycling between protozoan and metazoan hosts.

Staphylococcus aureus capsule synthesis requires the precursor N-acetyl-glucosamine; however, capsule is synthesized during post-exponential growth when the availability of N-acetyl-glucosamine is limited. Capsule biosynthesis also requires aerobic respiration, leading us to hypothesize that capsule synthesis requires tricarboxylic acid cycle intermediates. Consistent with this hypothesis, S. aureus tricarboxylic acid cycle mutants fail to make capsule.Staphylococcus aureus produces two major exopolysaccharides: poly-N-acetylglucosamine (PNAG) and capsule. PNAG is synthesized by enzymes encoded by genes within the intercellular adhesin (ica) operon (icaADBC) (5, 15) during the exponential growth phase when C 6 carbohydrates are abundant (5,10,12). In contrast, capsular polysaccharides are predominantly synthesized during the post-exponential growth phase when C 6 carbohydrates are in short supply (13). The most commonly encountered S. aureus capsule types, 5 and 8, are synthesized from the amino sugars . Interestingly, the biosynthetic precursor of the capsular sugars is UDP-N-acetylglucosamine (11), the same amino sugar used in synthesizing PNAG. N-acetylglucosamine is synthesized from the glycolytic intermediate fructose 6-phosphate, and in a rich medium containing glucose, abundant levels of fructose 6-phosphate will be generated by glycolysis. As stated, capsule is most abundantly synthesized in the post-exponential phase of growth when glucose is growth limiting (4, 13).In the absence of glucose, fructose 6-phosphate can be synthesized by gluconeogenesis from the tricarboxylic acid (TCA) cycle intermediate oxaloacetate. To do this, oxaloacetate undergoes an ATP-dependent decarboxylation and phosphorylation by phosphoenolpyruvate carboxykinase (pckA) to generate phosphoenolpyruvate (PEP) (19). Gluconeogenesis can then generate fructose 6-phosphate from PEP, which can be used for UDP-activated N-acetylglucosamine biosynthesis. Support for the idea that post-exponential-phase capsule biosynthesis requires TCA cycle activity and PEP carboxykinase can be found in the observation that capsule is made during aerobic growth (6). In the Dassy and Fournier study, respiratory chain inhibitors were used to show that respiratory activity or a high proton motive force is required for post-exponentialgrowth-phase capsule biosynthesis (6). Although this was an excellent study, it was unclear as to why inhibiting respiratory activity prevented capsule biosynthesis. One consequence of inhibiting respiratory activity is the accumulation of reducing potential in the form of NADH. As NADH accumulates, the intracellular concentration of NAD ϩ decreases and the activity of NAD ϩ -requiring enzymes similarly decreases. During the post-exponential growth phase, the primary consumer of NAD ϩ is the TCA cycle; therefore, inhibiting respiratory activity inhibits TCA cycle activity. Further support that postexponential capsule biosynthesis requires TCA cycle activity can be seen in transposon mutagenesis studies that found inactivation...

Legionella pneumophila uses a single homodimeric disulfide bond (DSB) oxidoreductase DsbA2 to catalyze extracytoplasmic protein folding and to correct DSB errors through protein-disulfide isomerase (PDI) activity. In Escherichia coli, these functions are separated to avoid futile cycling. In L. pneumophila, DsbA2 is maintained as a mixture of disulfides (S-S) and free thiols (SH), but when expressed in E. coli, only the SH form is observed. We provide evidence to suggest that structural differences in DsbB oxidases (LpDsbB1 and LpDsbB2) and DsbD reductases (LpDsbD1 and LpDsbD2) (compared to E. coli) permit bifunctional activities without creating a futile cycle. LpdsbB1 and LpdsbB2 partially complemented an EcdsbB mutant while neither LpdsbD1 nor LpdsbD2 complemented an EcdsbD mutant unless DsbA2 was also expressed. When the dsb genes of E. coli were replaced with those of L. pneumophila, motility was restored and DsbA2 was present as a mixture of redox forms. A dominant-negative approach to interfere with DsbA2 function in L. pneumophila determined that DSB oxidase activity was necessary for intracellular multiplication and assembly/function of the Dot/Icm Type IVb secretion system. Our studies show that a single-player system may escape the futile cycle trap by limiting transfer of reducing equivalents from LpDsbDs to DsbA2.