The Staphylococcus aureus Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by strains epidemiologically associated with the current outbreak of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and with the often-lethal necrotizing pneumonia. To investigate the role of PVL in pulmonary disease, we tested the pathogenicity of clinical isolates, isogenic PVL-negative and PVL-positive S. aureus strains, as well as purified PVL, in a mouse acute pneumonia model. Here we show that PVL is sufficient to cause pneumonia and that the expression of this leukotoxin induces global changes in transcriptional levels of genes encoding secreted and cell wall-anchored staphylococcal proteins, including the lung inflammatory factor staphylococcal protein A (Spa).

Previous reports indicated a correlation between loss of plasmids and decreased infectivity of Borrelia burgdorferi strain B31, suggesting that plasmids may encode proteins that are required for pathogenesis. In this study, we expand on this correlation. Using the B. burgdorferi genomic sequence, we designed primers specific for each plasmid, and by using PCR we catalogued 11 linear and 2 circular plasmids from 49 clonal isolates of a mid-passage B. burgdorferi strain B31, initially derived from infected mouse skin, and 20 clones obtained from mouse skin infected with a low-passage isolate of B. burgdorferi strain B31. Among the 69 clones analyzed, nine distinct genotypes were identified relative to wild-type B. burgdorferi strain B31. Among the nine clonal genotypes obtained, only the 9-kb circular plasmid (cp9), the 25-kb linear plasmid (lp25), and either the 28-kb linear plasmid 1 or 4 (lp28-1 and lp28-4, respectively) were missing, in different combinations. We compared the infectivity of the wild-type strain, containing all known B. burgdorferi plasmids, with those of single mutants lacking either lp28-1, lp28-4, or lp25 and a double mutant missing both cp9 and lp28-1. The infectivity data indicated that B. burgdorferi strain B31 cells lacking lp28-4 were modestly attenuated in all tissues analyzed, whereas samples missing lp25 were completely attenuated in all tissues, even at the highest inoculum tested. Isolates without lp28-1 infected the joint tissue yet were not able to infect other tissues as effectively. In addition, we have observed a selection in vivo in the skin, bladder, and joint for cells containing lp25 and in the skin and bladder for cells containing lp28-1, indicating that lp25 and lp28-1 encode proteins required for colonization and short-term maintenance in these mammalian tissues. In contrast, there was no selection in the joint for cells containing lp28-1, suggesting that genes on lp28-1 are not required for colonization of B. burgdorferi within the joint. These observations imply that the dynamic nature of the B. burgdorferi genome may provide the genetic heterogeneity necessary for survival in the diverse milieus that this pathogen occupies in nature and may contribute to tropism in certain mammalian host tissues.Lyme borreliosis, or Lyme disease, is a multisystemic inflammatory disorder transmitted by the bites of ticks infected with the spirochetal bacterium Borrelia burgdorferi sensu lato (13,25). Over the past 15 years, Lyme disease has become the most frequent arthropod-borne disease in the United States and has been identified in a latitudinal strip in the Northern hemisphere including Europe, Eurasia, and Asia (2, 4, 5, 29). Determination of the genomic sequence of B. burgdorferi strain B31 by The Institute of Genomic Research (TIGR), along with several other investigators, in 1997 revealed that this spirochete contained a unique genomic organization consisting of a linear chromosome of 910 kb in size and several plasmids (reference 8 and the TIGR website [http://www.tigr.org/t...

SummaryBorrelia burgdorferi , the aetiological agent of Lyme disease, utilizes multiple adhesins to interact with both the arthropod vector and mammalian hosts it colonizes. One such adhesive molecule is a surfaceexposed fibronectin-binding lipoprotein, designated BBK32. Previous characterization of BBK32-mediated fibronectin binding has been limited to biochemical analyses due to the difficulty in mutagenizing infectious isolates of B. burgdorferi . Here we report an alternative method to inactivate bbk32 via allelic exchange through use of a low-passage variant of B. burgdorferi strain B31 that is more readily transformed. The resulting mutant does not synthesize BBK32, exhibits reduced fibronectin binding in solid phase assays and manifests decreased interactions with mouse fibroblast cells relative to both the infectious parent and genetic complement. Furthermore, the bbk32 knockout was significantly attenuated in the murine model of Lyme disease, whereas a genetically complemented control was not, indicating that BBK32 is necessary for maximal B. burgdorferi infection in the mouse. To our knowledge this is the first mutational analysis of a surface exposed, functional borrelial lipoprotein adhesin whose activity is associated with the mammalian host environment. By analogy with other pathogens that utilize fibronectin binding as an important virulence determinant, the borrelial fibronectin-BBK32 interaction is likely to be important in B. burgdorferi-specific pathogenic mechanisms, particularly in the context of dissemination, secondary colonization and/or persistence.

The 25-kb linear plasmid lp25 and one of the 28-kb linear plasmids (lp28-1) are required for experimental infection in Borrelia burgdorferi, the etiologic agent of Lyme disease. The loss of these plasmids either eliminates infectivity (lp25) or significantly increases the 50% infective dose during a 2-week infection period (lp28-1). This study assessed the kinetics of bacterial dissemination in C3H/HeN mice infected with B. burgdorferi lacking either lp25 or lp28-1, as well as their wild-type parent, and tracked the development of specific borrelial antibodies over a 3-week period. The results indicated that the wild type and the lp28-1 ؊ strains were able to disseminate throughout the host, whereas the lp25 ؊ strain was cleared within 48 h of inoculation. While the wild-type B. burgdorferi persisted in tissues for the duration of the study, the lp28-1 ؊ mutant began clearing at day 8, with no detectable bacteria present by day 18. As expected, the wild-type strain persisted in C3H/HeN mice despite a strong humoral response; however, the lp28-1 ؊ mutant was cleared coincidently with the development of a modest immunoglobulin M response. The lp28-1 ؊ mutant was able to disseminate and persist in C3H-scid mice at a level indistinguishable from that of wild-type cells, confirming that acquired immunity was required for clearance in C3H/HeN mice. Thus, within an immunocompetent host, lp28-1-encoded proteins are not required for dissemination but are essential for persistence associated with Lyme borreliosis.Lyme disease or Lyme borreliosis is a multisystemic disorder transmitted by ticks of the genus Ixodes infected with the spirochetal bacterium Borrelia burgdorferi sensu lato, composed of B. burgdorferi sensu stricto, B. garinii, and B. afzelii (1,8,16). Lyme disease is the leading zoonotic infection in the United States, accounting for 95% of all reported vector-borne illness (10). The completed B. burgdorferi sensu stricto strain B31 annotated sequence revealed an unusual genome containing a linear chromosome of 910 kb in size as well as several extrachromosomal elements, including 12 linear plasmids of sizes ranging from 5 to 56 kb and 9 circular plasmids with sizes between 9 and 32 kb (9,14).Early studies had demonstrated a correlation between plasmid content and infectivity but were hindered by the plethora of plasmid species in B. burgdorferi and the inherent difficulty in resolving the many linear and circular species (24, 29-31). As such, prior to the information gained from the B. burgdorferi strain B31 genome sequence, it was difficult to definitively link a specific plasmid to a defect in infectivity. Exploiting the completed genome sequence of B. burgdorferi, we designed oligonucleotide primers that could be used in conjunction with PCR to specifically catalog the presence of plasmids from clonal isolates of B. burgdorferi (17). In our quest to link infectivity to any of the plasmids, we determined, along with others, that two linear plasmids, 25 and 28 kb in size (designated lp25 and lp28-1, respectiv...

The LspA1, LspA2, and LspB proteins of Haemophilus ducreyi comprise a two-partner secretion system that has been shown to be necessary for H. ducreyi to inhibit phagocytosis by immune cells in vitro. Inactivation of lspA1 resulted in increased levels of LspA2, suggesting that these two proteins are differentially controlled (C. J. Ward et al., Infect. Immun. 71:2478-2486, 2003). Expression of LspA2 but not LspA1 was shown to be both growth phase dependent and affected by the presence of fetal calf serum (FCS) in the growth medium. In addition, neither LspA1 nor LspA2 could be detected in culture supernatant fluid in the absence of FCS. DNA microarray analysis revealed that 324 H. ducreyi genes were differentially regulated after growth in the presence of FCS. Among these, the CpxRA two-component sensory transduction system was downregulated by the presence of FCS. Inactivation of cpxR resulted in increased expression of both LspB and LspA2. Electrophoretic mobility shift assays showed that a recombinant H. ducreyi CpxR protein bound the promoter region of the lspB-lspA2 operon. The cpxR and cpxA genes were shown to be part of an operon containing two additional genes in H. ducreyi 35000HP. This is the first description of a two-component sensory transduction system regulating a proven virulence factor of H. ducreyi.Haemophilus ducreyi is a gram-negative coccobacillus and the causative agent of the sexually transmitted genital ulcer disease (GUD) chancroid (1,8). Globally, chancroid is a significant sexually transmitted disease, with more than 6 million cases reported in 1997 (60). In the United States, several outbreaks were reported between 1980 and 2000 (24, 36, 51), but since then the number of cases has greatly diminished, and today the soft chancres characteristic of H. ducreyi infection occur only in isolated cases that are typically associated with the sex trade industry (57). Chancroid is endemic in some developing countries in Africa, Asia, and South America, where it accounts for almost half of all GUD cases, although these numbers could be higher as H. ducreyi cases remain poorly documented (55,57). GUD is a recognized cofactor for human immunodeficiency virus acquisition and transmission (25,37), and a better understanding of H. ducreyi pathogenesis is necessary to allow a rational approach to the identification of vaccine candidates that could be used to prevent chancroid.H. ducreyi is a strict human pathogen, and it is likely that during the different stages of ulcer production (i.e., progression of a papule into a pustule followed by frank ulceration [1,8,50]), this pathogen controls gene expression to enhance its growth and to evade the host immune system. Information about regulatory networks that might control the expression of H. ducreyi virulence factors is very limited at present. Although nucleotide sequence analysis of the H. ducreyi 35000HP genome (GenBank accession no. NC002940) revealed the presence of several genes encoding predicted proteins with homology to known bacterial regulators...

The Haemophilus ducreyi 35000HP genome encodes a homolog of the CpxRA two-component cell envelope stress response system originally characterized in Escherichia coli. CpxR, the cytoplasmic response regulator, was shown previously to be involved in repression of the expression of the lspB-lspA2 operon (M. LabandeiraRey, J. R. Mock, and E. J. Hansen, Infect. Immun. 77:3402-3411, 2009). In the present study, the H. ducreyi CpxR and CpxA proteins were shown to closely resemble those of other well-studied bacterial species. A cpxA deletion mutant and a CpxR-overexpressing strain were used to explore the extent of the CpxRA regulon. DNA microarray and real-time reverse transcriptase (RT) PCR analyses indicated several potential regulatory targets for the H. ducreyi CpxRA two-component regulatory system. Electrophoretic mobility shift assays (EMSAs) were used to prove that H. ducreyi CpxR interacted with the promoter regions of genes encoding both known and putative virulence factors of H. ducreyi, including the lspB-lspA2 operon, the flp operon, and dsrA. Interestingly, the use of EMSAs also indicated that H. ducreyi CpxR did not bind to the promoter regions of several genes predicted to encode factors involved in the cell envelope stress response. Taken together, these data suggest that the CpxRA system in H. ducreyi, in contrast to that in E. coli, may be involved primarily in controlling expression of genes not involved in the cell envelope stress response.

Numerous studies have provided suggestive evidence that the loss of plasmids correlates with the loss of infectivity of the Lyme disease spirochetes. In this study we have further investigated this correlation. Clonal populations were obtained from the skin of a mouse infected for 3 months with a clonal population of Borrelia burgdorferi B31MI. The complete plasmid compositions of these populations were determined using a combination of PCR and Southern hybridization. The infectivities of clones differing in plasmid composition were tested using the C3H-HeJ murine model for Lyme disease. While several clones were found to be noninfectious, a correlation between the loss of a specific plasmid and loss of infectivity in the clones analyzed in this report was not observed. While it is clear from recent studies that the loss of some specific plasmids results in attenuated virulence, this study demonstrates that additional mechanisms also contribute to the loss of infectivity.Infection with pathogenic species of the Borrelia burgdorferi sensu lato complex can lead to Lyme disease, an infection characterized by highly variable multisystem clinical manifestations (30, 31). Early studies suggested that plasmid-encoded proteins play an important role in Borrelia pathogenesis (26,28), and in recent years it has been demonstrated that they also play an important role in immune evasion (37,43). In addition, several plasmid-borne genes have been demonstrated to be up-regulated during infection, supporting a functional role for the proteins in the mammalian environment (1,11,39). The ability to define a correlation between specific plasmids and infectivity has until recently been complicated by the inherent instability of the Borrelia plasmids and the variation in plasmid content among isolates (3,9,17,18,20,25,28,29,40,41). Prior to the determination of the complete genome sequence and plasmid content of B. burgdorferi, these features made it difficult to interpret and compare much of the earlier data. Determination of the genome sequence of B. burgdorferi B31MI and other analyses revealed the existence of both linear and circular comigrating plasmids of between 24 and 56 kb that could not be distinguished by agarose gel electrophoresis (5, 10, 15, 35). There are also several distinct yet closely related circular plasmids (designated cp) of approximately 32 kb (cp32) (33, 35) and multiple linear plasmids (designated lp) of approximately 28 kb (lp28s). The cp32s have extended regions of homology, while the lp28s have significantly less identity. In addition to comigrating plasmids, the genome also contains an extraordinary number of plasmid-carried paralogous gene families (n ϭ 175). This genetic redundancy, which could allow for functional complementation of plasmid-encoded proteins, coupled with the inherent instability of the Borrelia plasmids has made it difficult to pinpoint one or more specific plasmids as essential for infectivity (4,9,17,28,37,38,43). However, two recent independent studies by Labandeira-Rey and Skare...

Pseudomonas aeruginosa is a highly virulent, multidrug-resistant pathogen that causes significant morbidity and mortality in hospitalized patients and is particularly devastating in patients with cystic fibrosis. Increasing antibiotic resistance coupled with decreasing numbers of antibiotics in the developmental pipeline demands novel antibacterial approaches. Here, we tested peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), which inhibit translation of complementary mRNA from specific, essential genes in P. aeruginosa. PPMOs targeted to acpP, lpxC, and rpsJ, inhibited P. aeruginosa growth in many clinical strains and activity of PPMOs could be enhanced 2-to 8-fold by the addition of polymyxin B nonapeptide at subinhibitory concentrations. The PPMO targeting acpP was also effective at preventing P. aeruginosa PAO1 biofilm formation and at reducing existing biofilms. Importantly, treatment with various combinations of a PPMO and a traditional antibiotic demonstrated synergistic growth inhibition, the most effective of which was the PPMO targeting rpsJ with tobramycin. Furthermore, treatment of P. aeruginosa PA103-infected mice with PPMOs targeting acpP, lpxC, or rpsJ significantly reduced the bacterial burden in the lungs at 24 h by almost 3 logs. Altogether, this study demonstrates that PPMOs targeting the essential genes acpP, lpxC, or rpsJ in P. aeruginosa are highly effective at inhibiting growth in vitro and in vivo. These data suggest that PPMOs alone or in combination with antibiotics represent a novel approach to addressing the problems associated with rapidly increasing antibiotic resistance in P. aeruginosa.