A live auxotrophic vaccine confers mucosal immunity and protection against lethal pneumonia caused by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the leading causes of nosocomial pneumonia and its associated mortality. Moreover, extensively drug-resistant high-risk clones are globally widespread, presenting a major challenge to the healthcare systems. Despite this, no vaccine is available against this high-concerning pathogen. Here we tested immunogenicity and protective efficacy of an experimental live vaccine against P. aeruginosa pneumonia, consisting of an auxotrophic strain which lacks the key enzyme involved in D-glutamate biosynthesis, a structural component of the bacterial cell wall. As the amounts of free D-glutamate in vivo are trace substances in most cases, blockage of the cell wall synthesis occurs, compromising the growth of this strain, but not its immunogenic properties. Indeed, when delivered intranasally, this vaccine stimulated production of systemic and mucosal antibodies, induced effector memory, central memory and IL-17A-producing CD4+ T cells, and recruited neutrophils and mononuclear phagocytes into the airway mucosa. A significant improvement in mice survival after lung infection caused by ExoU-producing PAO1 and PA14 strains was observed. Nearly one third of the mice infected with the XDR high-risk clone ST235 were also protected. These findings highlight the potential of this vaccine for the control of acute pneumonia caused by this bacterial pathogen.

Pleiotropic effects of cell wall amidase LytA on Streptococcus pneumoniae sensitivity to the host immune response.

The complement system is a key component of the host immune response for the recognition and clearance of Streptococcus pneumoniae. In this study, we demonstrate that the amidase LytA, the main pneumococcal autolysin, inhibits complement-mediated immunity independently of effects on pneumolysin by a complex process of impaired complement activation, increased binding of complement regulators, and direct degradation of complement C3. The use of human sera depleted of either C1q or factor B confirmed that LytA prevented activation of both the classical and alternative pathways, whereas pneumolysin inhibited only the classical pathway. LytA prevented binding of C1q and the acute-phase protein C-reactive protein to S. pneumoniae, thereby reducing activation of the classical pathway on the bacterial surface. In addition, LytA increased recruitment of the complement downregulators C4BP and factor H to the pneumococcal cell wall and directly cleaved C3b and iC3b to generate degradation products. As a consequence, C3b deposition and phagocytosis increased in the absence of LytA and were markedly enhanced for the lytA ply double mutant, confirming that a combination of LytA and Ply is essential for the establishment of pneumococcal pneumonia and sepsis in a murine model of infection. These data demonstrate that LytA has pleiotropic effects on complement activation, a finding which, in combination with the effects of pneumolysin on complement to assist with pneumococcal complement evasion, confirms a major role of both proteins for the full virulence of the microorganism during septicemia.

In vitro biofilm formation by Streptococcus pneumoniae as a predictor of post-vaccination emerging serotypes colonizing the human nasopharynx.

The increasing use of the 7-valent pneumococcal conjugate vaccine has been accompanied by the rise of non-vaccine serotypes colonizing the human nasopharynx. The vast majority of infections are caused by microorganisms that grow in biofilms. It has recently been shown that the formation of Streptococcus pneumoniae biofilms in vivo and in vitro is hindered by the presence of capsular polysaccharide. The biofilm-forming capacity of pneumococcal clinical isolates with different types of capsular polysaccharide and various isogenic transformants was examined. Strains of serotypes 19A and 19F, but not 19B and 19C, formed ≥ 80% of the quantity of biofilm associated with a non-encapsulated control strain. Strains of serogroup 6 also showed significant biofilm-forming capacity. The capsules of serotypes 19A and 19F, and serogroup 6 contain the disaccharides α-D-Glcp-(1→2)-α-L-Rhap-(1→ and α-D-Glcp-(1→3)-α-L-Rhap-(1→. Serotype 18A and serotypes 18B/18C have very similar capsular disaccharides: α-D-GlcpNAc-(1→3)-ß-L-Rhap-(1→ and α-D-Glcp-(1→3)-ß-L-Rhap-(1→ respectively. However, the strains of serogroup 18 showed impaired biofilm formation. These results indicate that the chemical composition/structure of the capsular polysaccharide is crucial to the biofilm-forming capacity of pneumococcal serotypes. Testing of the in vitro biofilm-forming ability of isogenic transformants expressing different capsular polysaccharides may help predict the emergence of colonizing, non-vaccine serotypes.

Frequent carriage of resistance mechanisms to ß-lactams and biofilm formation in Haemophilus influenzae causing treatment failure and recurrent otitis media in young children.

OBJECTIVES: Non-typeable Haemophilus influenzae are a major cause of acute otitis media (AOM), including chronic and recurrent otitis in young children. The objective of this study was to determine whether non-typeable H. influenzae isolates causing these infections produce biofilms and carry resistance mechanisms to ß-lactams. METHODS: A collection of 48 H. influenzae isolates was obtained by tympanocentesis or from otorrhoea samples from individual patients <3 years of age and diagnosed with recurrent or treatment failure AOM. Each isolate was surveyed for the presence of blaTEM genes, amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3) and biofilm formation in microtitre plates. RESULTS: In 43 of the 48 isolates (89.6%), at least one of the three tested conditions was identified: biofilm formation (83.3%) and resistance mechanisms to ß-lactams (33.3%), modifications in the transpeptidase domain of PBP3 being the most prevalent (22.9%), followed by ß-lactamase production (10.4%). Additionally, 13 (27.1%) isolates had two or more of these three traits. In relation to biofilm formation, those isolates with an amoxicillin MIC ≤ 0.5 mg/L had higher optical density values than isolates with an amoxicillin MIC ≥ 1 mg/L (Mann-Whitney U-test, P=0.048). CONCLUSIONS: These findings suggest that the successful treatment of non-typeable H. influenzae causing chronic and recurrent AOM in young children may be compromised by the high biofilm-forming capacity of the isolates and the presence of ß-lactam resistance mechanisms, particularly PBP3 mutations.

Biofilm formation avoids complement immunity and phagocytosis of Streptococcus pneumoniae.

Streptococcus pneumoniae is a frequent member of the microbiota of the human nasopharynx. Colonization of the nasopharyngeal tract is a first and necessary step in the infectious process and often involves the formation of sessile microbial communities by this human pathogen. The ability to grow and persist as biofilms is an advantage for many microorganisms, because biofilm-grown bacteria show reduced susceptibility to antimicrobial agents and hinder recognition by the immune system. The extent of host protection against biofilm-related pneumococcal disease has not been determined yet. Using pneumococcal strains growing as planktonic cultures or as biofilms, we have investigated the recognition of S. pneumoniae by the complement system and its interactions with human neutrophils. Deposition of C3b, the key complement component, was impaired on S. pneumoniae biofilms. In addition, binding of C-reactive protein and the complement component C1q to the pneumococcal surface was reduced in biofilm bacteria, demonstrating that pneumococcal biofilms avoid the activation of the classical complement pathway. In addition, recruitment of factor H, the downregulator of the alternative pathway, was enhanced by S. pneumoniae growing as biofilms. Our results also show that biofilm formation diverts the alternative complement pathway activation by a PspC-mediated mechanism. Furthermore, phagocytosis of pneumococcal biofilms was also impaired. The present study confirms that biofilm formation in S. pneumoniae is an efficient means of evading both the classical and the PspC-dependent alternative complement pathways the host immune system.

Insight into the composition of the intercellular matrix of Streptococcus pneumoniae biofilms.

Biofilm matrices consist of a mixture of extracellular polymeric substances synthesized in large part by the biofilm-producing microorganisms themselves. These matrices are responsible for the cohesion and three-dimensional architecture of biofilms. The present study demonstrates the existence of a matrix composed of extracellular DNA, proteins and polysaccharides in the biofilm formed by the human pathogen Streptococcus pneumoniae. Extracellular DNA, visualized by fluorescent labelling, was an important component of this matrix. The existence of DNA-protein complexes associated with bacterial aggregates and other polymers was hypothesized based on the unexpected DNA binding activity of lysozyme LytC, a novel moonlighting protein. Actually, a 25-amino-acid-long peptide derived from LytC (positions 408 and 432 of the mature LytC) was also capable of efficiently binding to DNA. Moreover, the presence of intercellular DNA-LytC protein complexes in pneumococcal biofilms was demonstrated by confocal laser scanning microscopy. Evidence of extracellular polysaccharide different from the capsule was obtained by staining with Calcofluor dye and four types of lectin conjugated to Alexa fluorophores, and by incubation with glycoside hydrolases. The presence of residues of Glcp(1→4) and GlcNAc(1→4) (in its deacetylated form) in the pneumococcal biofilm was confirmed by GC-MS techniques.

Cross-Protection against Acute Staphylococcus aureus Lung Infection in Mice by a D-Glutamate Auxotrophic Vaccine Candidate.

Staphylococcus aureus is regarded as a threatening bacterial pathogen causing invasive pneumonia in healthcare settings and in the community. The continuous emergence of multidrug resistant strains is narrowing the treatment options for these infections. The development of an effective S. aureus vaccine is, therefore, a global priority. We have previously developed a vaccine candidate, 132 ΔmurI Δdat, which is auxotrophic for D-glutamate, and protects against sepsis caused by S. aureus. In the present study, we explored the potential of this vaccine candidate to prevent staphylococcal pneumonia, by using an acute lung infection model in BALB/c mice. Intranasal inoculation of the vaccine strain yielded transitory colonization of the lung tissue, stimulated production of relevant serum IgG and secretory IgA antibodies in the lung and distal vaginal mucosa and conferred cross-protection to acute pneumonia caused by clinically important S. aureus strains. Although these findings are promising, additional research is needed to minimize dose-dependent toxicity for safer intranasal immunization with this vaccine candidate.

A highly-safe live auxotrophic vaccine protecting against disease caused by non-typhoidal Salmonella Typhimurium in mice.

BACKGROUND: Salmonella enterica serovar Typhimurium (S. Typhimurium) has become an important intestinal pathogen worldwide and is responsible for lethal invasive infections in populations at risk. There is at present an unmet need for preventive vaccines. METHODS: IRTA GN-3728 genome was sequenced by Illumina and d-glutamate and d-glutamate/d-alanine knockout-auxotrophs were constructed. They were characterized using electron microscopy, growth/viability curves, reversion analysis, and motility/agglutination assays. Their potential as vaccine candidates were explored using two BALB/c mouse models for Salmonella infections: a systemic and an intestinal inflammation. Clinical signs/body weight and survival were monitored, mucosal lactoferrin and specific/cross-reactive IgA/IgG were quantified by enzyme-linked-immunosorbent assays and bacterial shedding/burden in fecal/tissues were evaluated. RESULTS: The d-glutamate auxotroph, IRTA ΔmurI, is highly attenuated, immunogenic and fully protective against systemic infection. The IRTA ΔmurI Δalr ΔdadX double auxotroph, constructed to reinforce vaccine safety, showed a higher level of attenuation and was 100% effective against systemic disease. In the intestinal model, it proved to be safe, yielding a low-degree of mucosal inflammation, short-term shedding and undetectable invasiveness in the long-term, while eliciting cross-reactive fecal IgA/serum IgG against clinically relevant multidrug-resistant (MDR) S. Typhimurium strains. It also conferred protection against homologous oral challenge, and protected mice from local and extra-intestinal dissemination caused by one MDR strain responsible for an international outbreak of highly severe human infections. Additionally, oral vaccination promoted extended survival after lethal heterologous infection. CONCLUSION: This study yielded a very safe S. Typhimurium vaccine candidate that could be further refined for mucosal application against disease in humans.

Comparison of the in vivo efficacy of ceftaroline fosamil, vancomycin and daptomycin in a murine model of methicillin-resistant Staphylococcus aureus bacteraemia.

The need for alternative drugs to treat methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia has led to a focus on ceftaroline, for which clinical data remain scarce. Herein, the efficacy of ceftaroline fosamil for the treatment of experimental MRSA bacteraemia was compared with that of approved therapies. Five MRSA strains were tested in an immunocompetent BALB/c bacteraemia model. Serum pharmacokinetics of ceftaroline fosamil were determined using HPLC/MS Q-TOF. Two hours after infection with the MRSA strains, mice were administered 50 mg/kg of ceftaroline fosamil every 6 h, for 24 h. This regimen yielded a T>MIC of 61.5% for an MIC of 1 mg/L and proved efficacious against all strains, including an hVISA strain with non-susceptibility to daptomycin, as indicated by the reduction (mean ± s.d.) in log10 CFU/mL in blood of 2.34 ± 0.33 and log10 CFU/g in kidney of 2.08 ± 0.22. Similarly, treatment with daptomycin yielded a log reduction of 2.30 ± 0.60 in blood and 2.14 ± 0.31 in kidney. The decrease in bacterial density was less accentuated after treatment with vancomycin, which yielded 1.84 ± 0.73 and 1.95 ± 0.32 log reductions in blood and kidney, respectively. The results of the study showed that the efficacy of ceftaroline fosamil against MRSA bacteraemia in mice is not inferior to that of vancomycin and daptomycin, and indicated the potential use of ceftaroline fosamil against difficult-to-treat S. aureus bacteraemia. Considering these promising data, clinical trials should be conducted to ascertain the efficacy of the drug for treating bloodstream infections in humans.

Double Auxotrophy to Improve the Safety of a Live Anti-Pseudomonas aeruginosa Vaccine.

Pseudomonas aeruginosa is an opportunistic nosocomial pathogen that causes serious infections in the respiratory tract of immunocompromised or critically ill patients, and it is also a significant source of bacteremia. Treatment of these infections can be complicated due to the emergence of multidrug-resistant P. aeruginosa strains worldwide. Hence, the development of prophylactic vaccines is a priority for at-risk patients. We have previously developed a vaccine candidate with a single auxotrophy for D-glutamate, PAO1 ΔmurI, which protects against sepsis and acute pneumonia caused by P. aeruginosa. Given the paramount importance of safety in the development of live attenuated vaccines, we have improved the safety of the vaccine candidate by reducing the probability of a reversion to virulence by the inclusion of an additional auxotrophy for D-alanine. Single and double auxotrophs behaved in a similar manner in relation to the attenuation level, immunogenicity and protective efficacy, but the double auxotroph has the advantage of being more stable and safer as a candidate vaccine against respiratory infections caused by P. aeruginosa.

A New Live Auxotrophic Vaccine Induces Cross-Protection against Klebsiella pneumoniae Infections in Mice.

The development of a whole-cell vaccine from bacteria auxotrophic for D-amino acids present in the bacterial cell wall is considered a promising strategy for providing protection against bacterial infections. Here, we constructed a prototype vaccine, consisting of a glutamate racemase-deficient mutant, for preventing Klebsiella pneumoniae infections. The deletion mutant lacks the murI gene and requires exogenous addition of D-glutamate for growth. The results showed that the K. pneumoniae ΔmurI strain is attenuated and includes a favourable combination of antigens for inducing a robust immune response and conferring an adequate level of cross-protection against systemic infections caused by K. pneumoniae strains, including some hypervirulent serotypes with elevated production of capsule polysaccharide as well as multiresistant K. pneumoniae strains. The auxotroph also induced specific production of IL-17A and IFN-γ. The rapid elimination of the strain from the blood of mice without causing disease suggests a high level of safety for administration as a vaccine.

Vancomycin tolerance in clinical and laboratory Streptococcus pneumoniae isolates depends on reduced enzyme activity of the major LytA autolysin or cooperation between CiaH histidine kinase and capsular polysaccharide.

Vancomycin is frequently added to standard therapy for pneumococcal meningitis. Although vancomycin-resistant Streptococcus pneumoniae strains have not been isolated, reports on the emergence of vancomycin-tolerant pneumococci are a cause of concern. To date, the molecular basis of vancomycin tolerance in S. pneumoniae is essentially unknown. We examined two vancomycin-tolerant clinical isolates, i.e. a purported autolysin negative (LytA(-)), serotype 23F isolate (strain S3) and the serotype 14 strain 'Tupelo', which is considered a paradigm of vancomycin tolerance. S3 was characterized here as carrying a frameshift mutation in the lytA gene encoding the main pneumococcal autolysin. The vancomycin tolerance of strain S3 was abolished by transformation to the autolysin-proficient phenotype. The original Tupelo strain was discovered to be a mixture: a strain showing a vancomycin-tolerant phenotype (Tupelo_VT) and a vancomycin-nontolerant strain (Tupelo_VNT). The two strains differed only in terms of a single mutation in the ciaH gene present in the VT strain. Most interestingly, although the vancomycin tolerance of Tupelo_VT could be overcome by increasing the LytA dosage upon transformation by a multicopy plasmid or by externally adding the autolysin, we show that vancomycin tolerance in S. pneumoniae requires the simultaneous presence of a mutated CiaH histidine kinase and capsular polysaccharide.

In vitro destruction of Streptococcus pneumoniae biofilms with bacterial and phage peptidoglycan hydrolases.

Host- and phage-coded cell wall hydrolases have been used to fight Streptococcus pneumoniae growing as planktonic cells in vitro as well as in animal models. Until now, however, the usefulness of these enzymes in biofilm-grown pneumococci has gone untested. The antipneumococcal activity of different cell wall hydrolases produced by S. pneumoniae and a number of its phages was examined in an in vitro biofilm model. The major pneumococcal autolysin LytA, an N-acetylmuramoyl-l-alanine amidase, showed the greatest efficiency in disintegrating S. pneumoniae biofilms. The phage-encoded lysozymes Cpl-1 and Cpl-7 were also very efficient. Biofilms formed by the close pneumococcal relatives Streptococcus pseudopneumoniae and Streptococcus oralis were also destroyed by the phage endolysins but not by the S. pneumoniae autolysin LytA. A cooperative effect of LytA and Cpl-1 in the disintegration of S. pneumoniae biofilms was recorded.

Crystal structure of CbpF, a bifunctional choline-binding protein and autolysis regulator from Streptococcus pneumoniae.

Phosphorylcholine, a crucial component of the pneumococcal cell wall, is essential in bacterial physiology and in human pathogenesis because it binds to serum components of the immune system and acts as a docking station for the family of surface choline-binding proteins. The three-dimensional structure of choline-binding protein F (CbpF), one of the most abundant proteins in the pneumococcal cell wall, has been solved in complex with choline. CbpF shows a new modular structure composed both of consensus and non-consensus choline-binding repeats, distributed along its length, which markedly alter its shape, charge distribution and binding ability, and organizing the protein into two well-defined modules. The carboxy-terminal module is involved in cell wall binding and the amino-terminal module is crucial for inhibition of the autolytic LytC muramidase, providing a regulatory function for pneumococcal autolysis.

In vitro and in vivo efficacy of combinations of colistin and different endolysins against clinical strains of multi-drug resistant pathogens.

The emergence of multidrug resistant (MDR) pathogenic bacteria is jeopardizing the value of antimicrobials, which had previously changed the course of medical science. In this study, we identified endolysins ElyA1 and ElyA2 (GH108-PG3 family), present in the genome of bacteriophages Ab1051Φ and Ab1052Φ, respectively. The muralytic activity of these endolysins against MDR clinical isolates (Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae) was tested using the turbidity reduction assay. Minimal inhibitory concentrations (MICs) of endolysin, colistin and a combination of endolysin and colistin were determined, and the antimicrobial activity of each treatment was confirmed by time kill curves. Endolysin ElyA1 displayed activity against all 25 strains of A. baumannii and P. aeruginosa tested and against 13 out of 17 strains of K. pneumoniae. Endolysin ElyA2 did not display any such activity. The combined antimicrobial activity of colistin and ElyA1 yielded a reduction in the colistin MIC for all strains studied, except K. pneumoniae. These results were confirmed in vivo in G. mellonella survival assays and in murine skin and lung infection models. In conclusion, combining colistin (1/4 MIC) with the new endolysin ElyA1 (350 µg) enhanced the bactericidal activity of colistin in both in vitro and in vivo studies. This will potentially enable reduction of the dose of colistin used in clinical practice.

Versatility of the capsular genes during biofilm formation by Streptococcus pneumoniae.

Streptococcus pneumoniae forms part of the natural microbiota of the nasopharynx. For the pneumococcus to cause infection, colonization needs to occur and this process is mediated by adherence of bacteria to the respiratory epithelium. Although the capsular polysaccharide (CPS) of S. pneumoniae is known to be important for infection to occur, its role in colonization is controversial. Biofilm models are starting to emerge as a promising tool to investigate the role of CPS during nasopharyngeal carriage, which is the first step in the dissemination and initiation of a pneumococcal infection. Using a well-defined model system to analyse in vitro biofilm formation in pneumococcus, here we explore the molecular changes underlying the appearance of capsular mutants using type 3 S. pneumoniae cells. Spontaneous colony phase variants show promoter mutations, as well as duplications, deletions and point mutations in the cap3A gene, which codes for a UDP-glucose dehydrogenase (UDP-GlcDH). Increased biofilm-forming capacity could usually be correlated with a reduction both in colony size and in the relative amount of CPS present on the cell surface of each colony variant. However, a mutation in Cap3A Thr83Ile (a strictly conserved residue in bacterial UDP-GlcDHs) that resulted in very low CPS production also led to impaired biofilm formation. We propose that non-encapsulated mutants of pneumococcal type 3 strains are essentially involved in the initial stages (the attachment stage) of biofilm formation during colonization/pathogenesis.

Pneumococcal biofilms.

Over 60% of bacterial infections (and up to 80% of chronic infections) are currently considered to involve microbial growth in biofilms. This peculiar form of life poses an array of problems in human clinical practice, from infections associated with the implant of prosthetic devices and dental plaque formation to diseases such as cystic fibrosis, otitis media, and endocarditis. Biofilms are also at the basis of a variety of problems in industry. This report describes the biofilms produced by Streptococcus pneumoniae. This bacterium often colonizes the upper airways in humans as a normal commensal, yet it may spread to other areas of the body, causing otitis media, pneumonia, or invasive diseases such as bacteremia and meningitis. The capacity of S. pneumoniae to form biofilms had not been explored until recently. Several newly developed in vitro systems have allowed to test the capacity of S. pneumoniae to form biofilms, and to analyze the influence of several factors, including DNA and proteins-which play a role in the virulence of this "supergerm" in the formation and development of biofilms. In this brief review, we update the knowledge available on pneumococcal biofilm formation and the unusual features of this structure.

A D-Alanine auxotrophic live vaccine is effective against lethal infection caused by Staphylococcus aureus.

Staphylococcus aureus infections are becoming a major global health issue due to the rapid emergence of multidrug-resistant strains. Therefore, there is an urgent need to develop an effective vaccine to prevent and control these infections. In order to develop a universal immunization strategy, we constructed a mutant derivative of S. aureus 132 which lacks the genes involved in D-alanine biosynthesis, a structural component of cell wall peptidoglycan. This unmarked deletion mutant requires the exogenous addition of D-alanine for in vitro growth. The aim of this study was to examine the ability of this D-alanine auxotroph to induce protective immunity against staphylococcal infection. Our findings demonstrate that this deletion mutant is highly attenuated, elicits a protective immune response in mice and generates cross-reactive antibodies. Moreover, the D-alanine auxotroph was completely eliminated from the blood of mice after its intravenous or intraperitoneal injection. We determined that the protective effect was dependent on antibody production since the adoptive transfer of immune serum into naïve mice resulted in effective protection against S. aureus bacteremia. In addition, splenocytes from mice immunized with the D-alanine auxotroph vaccine showed specific production of IL-17A after ex vivo stimulation. We conclude that this D-alanine auxotroph protects mice efficiently against virulent staphylococcal strains through the combined action of antibodies and IL-17A, and therefore constitutes a promising vaccine candidate against staphylococcal disease, for which no licensed vaccine is available yet.

The Streptococcus pneumoniaeyefM-yoeB and relBE Toxin-Antitoxin Operons Participate in Oxidative Stress and Biofilm Formation.

Type II (proteic) toxin-antitoxin systems (TAs) are widely distributed among bacteria and archaea. They are generally organized as operons integrated by two genes, the first encoding the antitoxin that binds to its cognate toxin to generate a harmless protein⁻protein complex. Under stress conditions, the unstable antitoxin is degraded by host proteases, releasing the toxin to achieve its toxic effect. In the Gram-positive pathogen Streptococcus pneumoniae we have characterized four TAs: pezAT, relBE, yefM-yoeB, and phD-doc, although the latter is missing in strain R6. We have assessed the role of the two yefM-yoeB and relBE systems encoded by S. pneumoniae R6 by construction of isogenic strains lacking one or two of the operons, and by complementation assays. We have analyzed the phenotypes of the wild type and mutants in terms of cell growth, response to environmental stress, and ability to generate biofilms. Compared to the wild-type, the mutants exhibited lower resistance to oxidative stress. Further, strains deleted in yefM-yoeB and the double mutant lacking yefM-yoeB and relBE exhibited a significant reduction in their ability for biofilm formation. Complementation assays showed that defective phenotypes were restored to wild type levels. We conclude that these two loci may play a relevant role in these aspects of the S. pneumoniae lifestyle and contribute to the bacterial colonization of new niches.

Crystallization and preliminary X-ray diffraction studies of choline-binding protein F from Streptococcus pneumoniae.

Choline-binding protein F (CbpF) is a modular protein that is bound to the pneumococcal cell wall through noncovalent interactions with choline moieties of the bacterial teichoic and lipoteichoic acids. Despite being one of the more abundant proteins on the surface, along with the murein hydrolases LytA, LytB, LytC and Pce, its function is still unknown. CbpF has been crystallized using the hanging-drop vapour-diffusion method at 291 K. Diffraction-quality orthorhombic crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 49.13, b = 114.94, c = 75.69 A. A SAD data set from a Gd-HPDO3A-derivatized CbpF crystal was collected to 2.1 A resolution at the gadolinium L(III) absorption edge using synchrotron radiation.