Protein kinase C-delta inhibition is organ-protective, enhances pathogen clearance, and improves survival in sepsis.

Sepsis is a leading cause of morbidity and mortality in intensive care units. Previously, we identified Protein Kinase C-delta (PKCδ) as an important regulator of the inflammatory response in sepsis. An important issue in development of anti-inflammatory therapeutics is the risk of immunosuppression and inability to effectively clear pathogens. In this study, we investigated whether PKCδ inhibition prevented organ dysfunction and improved survival without compromising pathogen clearance. Sprague Dawley rats underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. Post-surgery, PBS or a PKCδ inhibitor (200µg/kg) was administered intra-tracheally (IT). At 24 hours post-CLP, there was evidence of lung and kidney dysfunction. PKCδ inhibition decreased leukocyte influx in these organs, decreased endothelial permeability, improved gas exchange, and reduced blood urea nitrogen/creatinine ratios indicating organ protection. PKCδ inhibition significantly decreased bacterial levels in the peritoneal cavity, spleen and blood but did not exhibit direct bactericidal properties. Peritoneal chemokine levels, neutrophil numbers, or macrophage phenotypes were not altered by PKCδ inhibition. Peritoneal macrophages isolated from PKCδ inhibitor-treated septic rats demonstrated increased bacterial phagocytosis. Importantly, PKCδ inhibition increased survival. Thus, PKCδ inhibition improved survival and improved survival was associated with increased phagocytic activity, enhanced pathogen clearance, and decreased organ injury.

Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other Identified Enterococcus faecalis Phage Endolysins.

ϕEf11 is a temperate Siphoviridae bacteriophage that infects strains of Enterococcus faecalis The ϕEf11 genome, encompassing 65 open reading frames (ORFs), is contained within 42,822 bp of DNA. Within this genome, a module of six lysis-related genes was identified. Based upon sequence homology, one of these six genes, ORF28, was predicted to code for an N-acetylmuramoyl-l-alanine amidase endolysin of 46.133 kDa, composed of 421 amino acids. The PCR-amplified ORF28 was cloned and expressed, and the resulting gene product was affinity purified to homogeneity. The purified protein was obtained from a fusion protein that exhibited a molecular mass of 72.5 kDa, consistent with a 46.1-kDa protein combined with a fused 26.5-kDa glutathione S-transferase tag. It produced rapid, profound lysis in E. faecalis populations and was active against 73 of 103 (71%) E. faecalis strains tested. In addition, it caused substantial destruction of E. faecalis biofilms. The lysin was quite stable, retaining its activity for three years in refrigerated storage, was stable over a wide range of pHs, and was unaffected by the presence of a reducing agent; however, it was inhibited by increasing concentrations of Ca2+ Liquid chromatography-mass spectrometry analysis of E. faecalis cell wall digestion products produced by the ORF28 endolysin indicated that the lysin acted as an N-acetylmuramidase, an endo-ß-N-acetylglucosaminidase, and an endopeptidase, rather than an N-acetylmuramoyl-l-alanine amidase. The ϕEf11 ORF28 lysin shared 10% to 37% amino acid identity with the lytic enzymes of all other characterized E. faecalis bacteriophages.IMPORTANCE The emergence of multidrug-resistant pathogenic microorganisms has brought increasing attention to the urgent need for the development of alternative antimicrobial strategies. One such alternative to conventional antibiotics employs lytic enzymes (endolysins) that are produced by bacteriophages in the course of lytic infection. During lytic infection by a bacteriophage, these enzymes hydrolyze the cell wall peptidoglycan, resulting in the lysis of the host cell. However, external endolysin application can result in lysis from without. In this study, we have cloned, expressed, purified, and characterized an endolysin produced by a bacteriophage infecting strains of Enterococcus faecalis The lysin is broadly active against most of the tested E. faecalis strains and exhibits multifunctional enzymatic specificities that differ from all other characterized endolysins produced by E. faecalis bacteriophages.

Diverted Total Synthesis of Carolacton-Inspired Analogs Yields Three Distinct Phenotypes in Streptococcus mutans Biofilms.

The oral microbiome is a dynamic environment inhabited by both commensals and pathogens. Among these is Streptococcus mutans, the causative agent of dental caries, the most prevalent childhood disease. Carolacton has remarkably specific activity against S. mutans, causing acid-mediated cell death during biofilm formation; however, its complex structure limits its utility. Herein, we report the diverted total synthesis and biological evaluation of a rationally designed library of simplified analogs that unveiled three unique biofilm phenotypes further validating the role of natural product synthesis in the discovery of new biological phenomena.

Bacteria adhere less to barbed monofilament than braided sutures in a contaminated wound model.

BACKGROUND: Previous studies have found fewer clinical infections in wounds closed with monofilament suture compared with braided suture. Recently, barbed monofilament sutures have shown improved strength and increased timesavings over interrupted braided sutures. However, the adherence of bacteria to barbed monofilament sutures and other commonly used suture materials is unclear. QUESTIONS/PURPOSES: We therefore determined: (1) the adherence of bacteria to five suture types including a barbed monofilament suture; (2) the ability to culture bacteria after gentle washing of each suture type; and (3) the pattern of bacterial adherence. METHODS: We created an experimental contaminated wound model using planktonic methicillin-resistant Staphylococcus aureus (MRSA). Five types of commonly used suture material were used: Vicryl™, Vicryl™ Plus, PDS™, PDS™ Plus, and Quill™. To determine adherence, we determined the number of bacteria removed from the suture by sequential washes. Sutures were plated to determine bacterial growth. Sutures were examined under confocal microscopy to determine adherence patterns. RESULTS: The barbed monofilament suture showed the least bacterial adherence of any suture material tested. Inoculated monofilament and barbed monofilament sutures placed on agar plates had less bacterial growth than braided suture, whereas antibacterial monofilament and braided sutures showed no growth. Confocal microscopy showed more adherence to braided suture than to the barbed monofilament or monofilament sutures. CONCLUSIONS: Barbed monofilament suture showed similar bacterial adherence properties to standard monofilament suture. CLINICAL RELEVANCE: Our findings suggest barbed monofilament suture can be substituted for monofilament suture, at the surgeon's discretion, without fear of increased risk of infection.

Nitrate Is an Environmental Cue in the Gut for Salmonella enterica Serovar Typhimurium Biofilm Dispersal through Curli Repression and Flagellum Activation via Cyclic-di-GMP Signaling.

Curli, a major component of the bacterial biofilms in the intestinal tract, activates pattern recognition receptors and triggers joint inflammation after infection with Salmonella enterica serovar Typhimurium. The factors that allow S. Typhimurium to disperse from biofilms and invade the epithelium to establish a successful infection during acute inflammation remain unknown. Here, we studied S. Typhimurium biofilms in vitro and in vivo to understand how the inflammatory environment regulates the switch between multicellular and motile S. Typhimurium in the gut. We discovered that nitrate generated by the host is an environmental cue that induces S. Typhimurium to disperse from the biofilm. Nitrate represses production of an important biofilm component, curli, and activates flagella via the modulation of intracellular cyclic-di-GMP levels. We conclude that nitrate plays a central role in pathogen fitness by regulating the sessile-to-motile lifestyle switch during infection. IMPORTANCE Recent studies provided important insight into our understanding of the role of c-di-GMP signaling and the regulation of enteric biofilms. Despite an improved understanding of how c-di-GMP signaling regulates S. Typhimurium biofilms, the processes that affect the intracellular c-di-GMP levels and the formation of multicellular communities in vivo during infections remain unknown. Here, we show that nitrate generated in the intestinal lumen during infection with S. Typhimurium is an important regulator of biofilm formation in vivo.

The pdh operon is expressed in a subpopulation of stationary-phase bacteria and is important for survival of sugar-starved Streptococcus mutans.

Streptococcus mutans is a facultative member of the oral plaque and is associated with dental caries. It is able to survive long periods of sugar starvation. We show here that inactivation of pdhD, putatively encoding a subunit of the pyruvate dehydrogenase complex, impairs survival of both batch cultures and biofilms. We show that pdhD and the downstream genes pdhA, pdhB, and pdhC form an operon that is predominantly transcribed in stationary phase. Analysis with fluorescent reporters revealed a bimodal expression pattern for the pdh promoter, with less than 1% of stationary-phase populations expressing pdh. When it was first detected, after 1 day of sugar starvation in batch culture, expression was mostly in individual bacteria. At later times, expressing bacteria were often in chains. The lengths of the chains increased with time. We infer that the pdh-expressing subpopulation is able grow and divide and to persist for extended times in stationary phase.

Enhanced eradication of intracellular and biofilm-residing methicillin-resistant Staphylococcus aureus (MRSA) reservoirs with hybrid nanoparticles delivering rifampicin.

Osteomyelitis carries a high risk of recurrence even after extended, aggressive antibiotic therapy. One of the key challenges is to eradicate the reservoirs of methicillin-resistant Staphylococcus aureus (MRSA) inside the host bone cells and their biofilms. Our goal is to develop rifampicin loaded lipid-polymer hybrid nanocarriers (Rf-LPN) and evaluate if they can achieve enhanced rifampicin delivery to eradicate these intracellular and biofilm-residing MRSA. After optimization of the composition, Rf-LPN demonstrated size around 110 nm in diameter that remained stable in serum-supplemented medium, drug payload up to 11.7% and sustained rifampicin release for 2 weeks. When comparing Rf-LPN with free rifampicin, moderate but significant (p < 0.05) improvement of the activities against three osteomyelitis-causing bacteria (USA300-0114, CDC-587, RP-62A) in planktonic form were observed. In comparison, the enhancements in the activities against the biofilms and intracellular MRSA by Rf-LPN were even more substantial. The MBEC50 values against USA300-0114, CDC-587, and RP-62A were 42 vs 155, 70 vs 388, and 265 ng/ml vs over 400 ng/ml, respectively, and up to 18.5-fold reduction in the intracellular MRSA counts in osteoblasts was obtained. Confocal microscope images confirmed extensive accumulation of Rf-LPN inside the biofilm matrix and MRSA-infected osteoblasts. Overall, in this proof-of-concept study we have developed and validated the strategy to exploit the nanoparticle-cell and nanoparticle-biofilm interactions with a new rifampicin nanoformulation for prevention of osteomyelitis recurrence and chronicity caused by the elusive MRSA.

Lipid-polymer hybrid nanoparticles carrying linezolid improve treatment of methicillin-resistant Staphylococcus aureus (MRSA) harbored inside bone cells and biofilms.

Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent pathogen causing osteomyelitis. The tendency of MRSA to evade standard antibiotic treatment by hiding inside bone cells and biofilms is a major cause of frequent osteomyelitis recurrence. In this study, we developed a lipid-polymer hybrid nanoparticle loading the antibiotic linezolid (LIN-LPN), and focused on evaluating if this new nanoantibiotic can achieve significant in vitro activities against these intracellular and biofilm-embedded MRSA. The optimal LIN-LPN formulation demonstrated both high linezolid payload (12.0% by weight of nanoparticles) and controlled release characteristics (gradually released the entrapped antibiotic in 120 h). Although it achieved lower activities against bacteria including USA300-0114, CDC-587, RP-62A in planktonic form, it was substantially superior against the intracellular MRSA reservoir inside osteoblast cells. The differences of intracellular activities between LIN-LPN and linezolid were 87.0-fold, 12.3-fold, and 12.6-fold in CFU/ml (p < 0.05 or < 0.01) at 2 µg/ml, 4 µg/ml, and 8 µg/ml linezolid concentrations, respectively. LIN-LPN also suppressed the MRSA biofilm growth to 35-60% of the values achieved with free linezolid (p < 0.05). These enhanced intracellular and anti-biofilm activities of LIN-LPN were likely contributed by the extensive accumulation of LIN-LPN inside the MRSA-infected osteoblasts and biofilms as revealed in the confocal microscope images. The study thus validates the feasibility of exploiting the good nanoparticle-host cell and nanoparticle-biofilm interactions for improving the antibiotic drug activities against the poorly accessible bacteria, and supports LIN-LPN as a new alternative therapy for preventing the recurrence of MRSA-mediated bone infections.

Salmonella Typhimurium biofilm disruption by a human antibody that binds a pan-amyloid epitope on curli.

Bacterial biofilms, especially those associated with implanted medical devices, are difficult to eradicate. Curli amyloid fibers are important components of the biofilms formed by the Enterobacteriaceae family. Here, we show that a human monoclonal antibody with pan-amyloid-binding activity (mAb 3H3) can disrupt biofilms formed by Salmonella enterica serovar Typhimurium in vitro and in vivo. The antibody disrupts the biofilm structure, enhancing biofilm eradication by antibiotics and immune cells. In mice, 3H3 injections allow antibiotic-mediated clearance of catheter-associated S. Typhimurium biofilms. Thus, monoclonal antibodies that bind a pan-amyloid epitope have potential to prevent or eradicate bacterial biofilms.

Role of intracellular polysaccharide in persistence of Streptococcus mutans.

Intracellular polysaccharide (IPS) is accumulated by Streptococcus mutans when the bacteria are grown in excess sugar and can contribute toward the cariogenicity of S. mutans. Here we show that inactivation of the glgA gene (SMU1536), encoding a putative glycogen synthase, prevented accumulation of IPS. IPS is important for the persistence of S. mutans grown in batch culture with excess glucose and then starved of glucose. The IPS was largely used up within 1 day of glucose starvation, and yet survival of the parental strain was extended by at least 15 days beyond that of a glgA mutant; potentially, some feature of IPS metabolism distinct from providing nutrients is important for persistence. IPS was not needed for persistence when sucrose was the carbon source or when mucin was present.

Generation of metabolically diverse strains of Streptococcus pyogenes during survival in stationary phase.

Streptococcus pyogenes, in addition to causing fulminant disease, can be carried asymptomatically and may survive in the host without causing disease. Long-term stationary-phase cultures were used to characterize the metabolism of cultures surviving after glucose depletion. Survival of stationary-phase cultures in glucose-depleted rich medium was truncated by switching the cells to phosphate-buffered saline or by the addition of antibiotics, suggesting that survival depended on the presence of nutrients and metabolic activity. The metabolites of the pyruvate-to-acetate (PA) pathway (acetate and formate) and amino acid catabolic pathways (ammonia) accumulated throughout long-term stationary phase (12 weeks). Acid and ammonia production was balanced so that the culture pH was maintained above pH 5.6. Strains isolated from long-term stationary-phase cultures accumulated mutations that resulted in unique exponential-phase metabolisms, with some strains expressing the PA pathway, some strains producing ammonia, and some strains expressing both in the presence of glucose. Strains expressing high levels of PA pathway activity during exponential growth were unable to survive when regrown in pure culture due to the production of excess acid. These data suggest that S. pyogenes diversifies during survival in stationary phase into distinct strains with different metabolisms and that complementary metabolism is required to control the pH in stationary-phase cultures. One of three survivor strains isolated from tonsillar discard material from patients expressed high levels of the PA pathway during exponential growth. Sequencing of multiple group A streptococcus regulators revealed two different mutations in two different strains, suggesting that random mutation occurs during survival.

Oxymetazoline solutions inhibit middle ear pathogens and are not ototoxic.

OBJECTIVES: This study was performed to explore the antimicrobial activity of two commercially available oxymetazoline hydrochloride preparations against the common pathogens of otitis media and to demonstrate the lack of ototoxicity of these agents and of United States Pharmacopeia (USP) oxymetazoline in a standard animal model. METHODS: Disc diffusion assays and minimum inhibitory concentration studies against American Type Culture Collection reference strains of common middle ear pathogens were used to evaluate the antimicrobial activity of oxymetazoline solutions and fluoroquinolone drops, and outer hair cell counts were performed on scanning electron micrographs of guinea pig basal cochlear segments after chronic exposure to oxymetazoline solutions and positive and negative controls. RESULTS: Oxymetazoline nasal spray and eyedrops had activity against all species tested except Haemophilus influenzae and Pseudomonas aeruginosa. The USP oxymetazoline had limited antimicrobial activity. Oxymetazoline nasal spray, oxymetazoline eyedrops, and USP oxymetazoline had ototoxicity profiles indistinguishable from that of the saline solution control. CONCLUSIONS: Commercially available oxymetazoline solutions are active against several of the common pathogens of otitis media. This antimicrobial activity is not due to oxymetazoline, and is more likely due to preservatives present in the solutions. The solutions tested are not ototoxic to guinea pig outer hair cells. Oxymetazoline solutions are potential substitutes for broad-spectrum antibiotic drops after tympanostomy tube placement.

Bioorganic Investigation of Multicationic Antimicrobials to Combat QAC-Resistant Staphylococcus aureus.

Quaternary ammonium compounds (QACs) have historically served as a first line of defense against pathogenic bacteria. Recent reports have shown that QAC resistance is increasing at an alarming rate, especially among methicillin-resistant Staphylococcus aureus (MRSA), and preliminary work has suggested that the number of cations present in the QAC scaffold inversely correlates with resistance. Given our interest in multiQACs, we initiated a multipronged approach to investigate their biofilm eradication properties, antimicrobial activity, and the propensity of methicillin-susceptible S. aureus (MSSA) to develop resistance toward these compounds. Through these efforts we identified multiQACs with superior profiles against resistant (MRSA) planktonic bacteria and biofilms. Furthermore, we document the ability of MSSA to develop resistance to several commercial monoQAC disinfectants and a novel aryl bisQAC, yet we observe no resistance to multiQACs. This work provides insight into the mechanism and rate of resistance development of MSSA and MRSA toward a range of QAC structures.

Mucin can enhance growth, biofilm formation, and survival of Streptococcus mutans.

Streptococcus mutans is a member of the dental plaque and is the primary causative agent of dental caries. It can survive extended periods of starvation, which may occur in different niches within the oral cavity. We have found that mucin compensated for the absence of amino acids to promote exponential growth and biofilm formation of S. mutans in minimal medium supplemented with glucose and sucrose, respectively. Mucin extended survival in conditions where there was no net growth provided the operon encoding the pyruvate dehydrogenase complex was intact. Mucin extended survival in conditions of amino acid sufficiency provided the tagatose pathway for galactose utilization was intact, suggesting that S. mutans can scavenge sufficient galactose from mucin to enhance survival, although not to serve as a primary carbon and energy source. The results suggest that mucin has a metabolic role in promoting survival of S. mutans.

Community-associated methicillin-resistant Staphylococcus aureus survival on artificial turf substrates.

OBJECTIVE: Artificial turf has been suggested as a risk factor for community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). This is an experimental study looking at survival of CA-MRSA on artificial turf. METHODS: MRSA strain USA-300-0114 was grown as either planktonic cells or biofilms in liquid cultures of beef heart infusion broth overnight at 37 °C. Beakers containing ProGrass (Pittsburgh, PA) turf were inoculated at the dirt interface with either ∼5 × 10 planktonic bacteria or with biofilms. The inoculum included varying nutrient conditions consisting of spent medium, saline, or 5% mucin. The beakers were incubated at 37 °C in ambient air. The main outcome measure was the number of surviving colony-forming units determined by plating on mannitol salt agar. RESULTS: Survival was biphasic with a colony-forming unit drop from ∼5 × 10 to ∼5 × 10 after the first week followed by survival of between 10 and 10 bacteria until termination of the experiment (20-50 d). Survival was dependent on nutrients, and washed cells survived less than 1 d. Mucin could serve as a nutrient source and slightly increased surviving numbers to 10-10 bacteria. Biofilm formation did not influence survival. CONCLUSIONS: CA-MRSA survivability on artificial turf surfaces is dependent on the availability of nutrients. These results suggest that CA-MRSA could survive on artificial turf in significant numbers for 1 wk, and lower numbers for at least 1 month, if supplied with appropriate nutrients. Outdoor environmental conditions may affect these findings.

Characterization of bacterial biofilms on tracheostomy tubes.

OBJECTIVES/HYPOTHESIS: To characterize the structure and microbial content of biofilms found on tracheostomy tubes. To determine the correlation between the patients' clinical condition and biofilm content. STUDY DESIGN: Prospective observational series. METHODS: Tracheostomy tubes were collected from patients in both the inpatient and outpatient setting at an urban academic medical center. Sections of the tracheostomy tubes were evaluated by confocal microscopy and bacteria from them plated and identified. The number of colony forming units (CFUs) and species present were determined and a univariate analysis performed to correlate them with various clinical factors. RESULTS: Bacteria were cultured from 19 of the 21 tracheostomy tubes collected. There were between 1 x 10(6) and 1 x 10(10) CFUs present in each of the 2 mm sections. Twelve different bacterial species and one fungus were isolated from culture and speciation. The number of bacteria isolated and the CFUs calculated varied in tubes obtained from the same patient at different times. CONCLUSIONS: Biofilms were present on tracheostomy tubes in greater than 90% of tracheostomy tubes collected as early as 7 days after insertion in both the inpatients and outpatients. Although a variety of bacteria were identified in the biofilm, they often appeared as discrete microcolonies that appeared to be monospecies biofilm on confocal microscopy. There was a statistically significant inverse correlation between the number of colony forming units found and frequency of inner cannula change.

Persistence of Streptococcus pyogenes in stationary-phase cultures.

In addition to causing fulminant disease, Streptococcus pyogenes may be asymptomatically carried between recurrent episodes of pharyngitis. To better understand streptococcal carriage, we characterized in vitro long-term stationary-phase survival (>4 weeks) of S. pyogenes. When grown in sugar-limited Todd-Hewitt broth, S. pyogenes cells remained culturable for more than 1 year. Both Todd-Hewitt supplemented with excess glucose and chemically defined medium allowed survival for less than 1 week. After 4 weeks of survival in sugar-limited Todd-Hewitt broth, at least 10(3) CFU per ml remained. When stained with fluorescent live-dead viability stain, there were a number of cells with intact membranes that were nonculturable. Under conditions that did not support persistence, these cells disappeared 2 weeks after loss of culturability. In persistent cultures, these may be cells that are dying during cell turnover. After more than 4 weeks in stationary phase, the culturable cells formed two alternative colony phenotypes: atypical large colonies and microcolonies. Protein expression in two independently isolated microcolony strains, from 14-week cultures, was examined by use of two-dimensional electrophoresis. The proteomes of these two strains exhibited extensive changes compared to the parental strain. While some of these changes were common to the two strains, many of the changes were unique to a single strain. Some of the common changes were in metabolic pathways, suggesting a possible alternate metabolism for the persisters. Overall, these data suggest that under certain in vitro conditions, S. pyogenes cells can persist for greater than 1 year as a dynamic population.

Persistence of Streptococcus mutans in stationary-phase batch cultures and biofilms.

Streptococcus mutans is a member of oral plaque biofilms and is considered the major etiological agent of dental caries. We have characterized the survival of S. mutans strain UA159 in both batch cultures and biofilms. Bacteria grown in batch cultures in a chemically defined medium, FMC, containing an excess of glucose or sucrose caused the pH to decrease to 4.0 at the entry into stationary phase, and they survived for about 3 days. Survival was extended up to 11 days when the medium contained a limiting concentration of glucose or sucrose that was depleted by the time the bacteria reached stationary phase. Sugar-limited cultures maintained a pH of 7.0 throughout stationary phase. Their survival was shortened to 3 days by the addition of exogenous lactic acid at the entry into stationary phase. Sugar starvation did not lead to comparable survival in biofilms. Although the pH remained at 7.0, bacteria could no longer be cultured from biofilms 4 days after the imposition of glucose or sucrose starvation; BacLight staining results did not agree with survival results based on culturability. In both batch cultures and biofilms, survival could be extended by the addition of 0.5% mucin to the medium. Batch survival increased to an average of 26 (+/-8) days, and an average of 2.7 x 10(5) CFU per chamber were still present in biofilms that were starved of sucrose for 12 days.

Involvement of Lsp, a member of the LraI-lipoprotein family in Streptococcus pyogenes, in eukaryotic cell adhesion and internalization.

Three open reading frames (ORFs) were identified by a genome walking strategy in the genomes of serotype M49 group A streptococcal (GAS) strains CS101 and 591. These ORFs were located between the mga core regulon and the dipeptide permease operon. The deduced amino acid (aa) sequences contained signature sequences indicative of a lipoprotein (306 aa), an intracellular protein (823 aa), and a secreted peptide (66 aa), respectively. ORF1 (named Lsp for lipoprotein of Streptococcus pyogenes) and ORF2 exhibited a high degree of homology to the lmb/ORF2 genes of S. agalactiae (B. Spellerberg et al., Infect. Immun. 67:871-878, 1999). The three ORFs were found to be present in each of the 27 GAS serotype strains tested. Transcription analysis revealed a polycistronic lsp/ORF2 and a monocistronic ORF3 message that were detected primarily at the transition from exponential to stationary growth phase. lsp and ORF2 mutants, ORF2- and ORF3-luciferase reporter fusions, and antiserum against recombinant Lsp were produced to examine the biological role of these genes. Although high Zn(2+) and Cu(2+) ion concentrations decreased lsp operon expression, Lsp did not transport divalent cations as described for other LraI-type operons. The lsp mutant had reduced fibronectin binding. Although no direct binding of Lsp to fibronectin could be demonstrated, the lsp mutant showed decreased transcription of prtF2 encoding the fibronectin-binding protein F2. Both the lsp and ORF2 mutants showed decreased laminin binding. Adherence to and internalization into A549 epithelial cells of both mutants was reduced without a detectable effect on eukaryotic cell viability. The transcription of a number of virulence factors was altered in the lsp mutants and ORF2 mutants. The changes in laminin binding and eukaryotic cell internalization could be explained by changes in transcription of speB (cysteine protease) and/or the global regulators mga, csrRS, and nra.