François Malouin

Bacterial c-di-GMP is an immunostimulatory molecule.

Cyclic diguanylate (c-di-GMP) is a bacterial intracellular signaling molecule. We have shown that treatment with exogenous c-di-GMP inhibits Staphylococcus aureus infection in a mouse model. We now report that c-di-GMP is an immodulator and immunostimulatory molecule. Intramammary treatment of mice with c-di-GMP 12 and 6 h before S. aureus challenge gave a protective effect and a 10,000-fold reduction in CFUs in tissues (p < 0.001). Intramuscular vaccination of mice with c-di-GMP coinjected with S. aureus clumping factor A (ClfA) Ag produced serum with significantly higher anti-ClfA IgG Ab titers (p < 0.001) compared with ClfA alone. Intraperitoneal injection of mice with c-di-GMP activated monocyte and granulocyte recruitment. Human immature dendritic cells (DCs) cultured in the presence of c-di-GMP showed increased expression of costimulatory molecules CD80/CD86 and maturation marker CD83, increased MHC class II and cytokines and chemokines such as IL-12, IFN-γ, IL-8, MCP-1, IFN-γ-inducible protein 10, and RANTES, and altered expression of chemokine receptors including CCR1, CCR7, and CXCR4. c-di-GMP-matured DCs demonstrated enhanced T cell stimulatory activity. c-di-GMP activated p38 MAPK in human DCs and ERK phosphorylation in human macrophages. c-di-GMP is stable in human serum. We propose that cyclic dinucleotides like c-di-GMP can be used clinically in humans and animals as an immunomodulator, immune enhancer, immunotherapeutic, immunoprophylactic, or vaccine adjuvant.

Plant antimicrobial agents and their effects on plant and human pathogens.

To protect themselves, plants accumulate an armoury of antimicrobial secondary metabolites. Some metabolites represent constitutive chemical barriers to microbial attack (phytoanticipins) and others inducible antimicrobials (phytoalexins). They are extensively studied as promising plant and human disease-controlling agents. This review discusses the bioactivity of several phytoalexins and phytoanticipins defending plants against fungal and bacterial aggressors and those with antibacterial activities against pathogens affecting humans such as Pseudomonas aeruginosa and Staphylococcus aureus involved in respiratory infections of cystic fibrosis patients. The utility of plant products as “antibiotic potentiators” and “virulence attenuators” is also described as well as some biotechnological applications in phytoprotection.

Novel riboswitch ligand analogs as selective inhibitors of guanine-related metabolic pathways.

Riboswitches are regulatory elements modulating gene expression in response to specific metabolite binding. It has been recently reported that riboswitch agonists may exhibit antimicrobial properties by binding to the riboswitch domain. Guanine riboswitches are involved in the regulation of transport and biosynthesis of purine metabolites, which are critical for the nucleotides cellular pool. Upon guanine binding, these riboswitches stabilize a 5′-untranslated mRNA structure that causes transcription attenuation of the downstream open reading frame. In principle, any agonistic compound targeting a guanine riboswitch could cause gene repression even when the cell is starved for guanine. Antibiotics binding to riboswitches provide novel antimicrobial compounds that can be rationally designed from riboswitch crystal structures. Using this, we have identified a pyrimidine compound (PC1) binding guanine riboswitches that shows bactericidal activity against a subgroup of bacterial species including well-known nosocomial pathogens. This selective bacterial killing is only achieved when guaA, a gene coding for a GMP synthetase, is under the control of the riboswitch. Among the bacterial strains tested, several clinical strains exhibiting multiple drug resistance were inhibited suggesting that PC1 targets a different metabolic pathway. As a proof of principle, we have used a mouse model to show a direct correlation between the administration of PC1 and the reduction of Staphylococcus aureus infection in mammary glands. This work establishes the possibility of using existing structural knowledge to design novel guanine riboswitch-targeting antibiotics as powerful and selective antimicrobial compounds. Particularly, the finding of this new guanine riboswitch target is crucial as community-acquired bacterial infections have recently started to emerge.

Binding of ceftaroline to penicillin-binding proteins of Staphylococcus aureus and Streptococcus pneumoniae

OBJECTIVES This study evaluated the affinity of ceftaroline and comparator beta-lactams for penicillin-binding proteins (PBPs) of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA) and Streptococcus pneumoniae with varying susceptibility to penicillin. Ceftaroline is currently in Phase 3 development for the treatment of complicated skin and skin structure infections and community-acquired pneumonia, including infections caused by MRSA and multidrug-resistant S. pneumoniae. METHODS Binding affinities (IC(50)s) of ceftaroline, ceftriaxone, oxacillin and penicillin G for PBPs were measured in a competition assay by adding various concentrations of the test drugs to membranes or whole cells. PBPs were labelled using the fluorescent reporter molecule Bocillin FL. RESULTS Overall, ceftaroline exhibited greater binding affinity for the range of PBPs tested, as compared with comparator beta-lactams. The high affinity of ceftaroline for PBPs 1-3 of MSSA and PBP2a of MRSA correlates well with its efficacy against these organisms, as determined by MIC. Similarly, efficient binding of ceftaroline to key S. pneumoniae PBPs, such as PBP2x/2a/2b, taken together, correlates well with its low MICs for penicillin-resistant isolates of S. pneumoniae. CONCLUSIONS The high affinities of ceftaroline for MRSA PBP2a, MSSA PBPs 1-3 and S. pneumoniae PBP2x/2a/2b support the potential efficacy of ceftaroline in the treatment of infections caused by MRSA and S. pneumoniae.

Transcription of Virulence Factors in Staphylococcus aureus Small-Colony Variants Isolated from Cystic Fibrosis Patients Is Influenced by SigB

Staphylococcus aureus small-colony variants (SCVs) are believed to account in part for the persistence of S. aureus during chronic infections. Little is understood about the gene expression profile that may explain the phenotype and distinguish SCVs from prototype S. aureus strains. In this study, DNA array transcriptional profiles of clinical SCVs isolated from the airways of cystic fibrosis patients were obtained and compared to those obtained from a laboratory-derived SCV strain (i.e., a respiratory-deficient hemB mutant) and prototype S. aureus strains. The genes commonly up-regulated in both hemB and clinical SCVs were found to be implicated in fermentation and glycolysis pathways. The well-known virulence regulator agr was not activated in SCVs, and such strains had low levels of alpha-toxin (hla) gene expression. Clinical SCVs also had a transcriptional signature of their own. Of striking interest is that many genes, most of them under the positive control of the alternate sigma factor SigB, were specifically up-regulated and differed in that way from that seen in prototype S. aureus and the hemB mutant. Since SigB influences up-regulation of adhesin type genes while indirectly down-regulating exoproteins and toxins, we evaluated the internalization and persistence of SCVs in mammalian cells. Results showed that clinical SCVs persisted much more efficiently in cells than the hemB and prototype strains and that a sigB mutant was a poor persister. Thus, it appears that the agr locus plays a minor role in the regulation of the virulon of SCVs, unlike SigB, which may have a key role in intracellular persistence.

Staphylococcus aureus sigma B-dependent emergence of small-colony variants and biofilm production following exposure to Pseudomonas aeruginosa 4-hydroxy-2-heptylquinoline-N-oxide

BackgroundStaphylococcus aureus and Pseudomonas aeruginosa are often found together in the airways of cystic fibrosis (CF) patients. It was previously shown that the P. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline-N- oxide (HQNO) suppresses the growth of S. aureus and provokes the emergence of small-colony variants (SCVs). The presence of S. aureus SCVs as well as biofilms have both been associated with chronic infections in CF.ResultsWe demonstrated that HQNO stimulates S. aureus to form a biofilm in association with the formation of SCVs. The emergence of SCVs and biofilm production under HQNO exposure was shown to be dependent on the activity of the stress- and colonization-related alternative sigma factor B (SigB). Analysis of gene expression revealed that exposure of a prototypical S. aureus strain to HQNO activates SigB, which was leading to an increase in the expression of the fibronectin-binding protein A and the biofilm-associated sarA genes. Conversely, the quorum sensing accessory gene regulator (agr) system and the α-hemolysin gene were repressed by HQNO. Experiments using culture supernatants from P. aeruginosa PAO1 and a double chamber co-culture model confirmed that P. aeruginosa stimulates biofilm formation and activates SigB in a S. aureus strain isolated from a CF patient. Furthermore, the supernatant from P. aeruginosa mutants unable to produce HQNO induced the production of biofilms by S. aureus to a lesser extent than the wild-type strain only in a S. aureus SigB-functional background.ConclusionsThese results suggest that S. aureus responds to HQNO from P. aeruginosa by forming SCVs and biofilms through SigB activation, a phenomenon that may contribute to the establishment of chronic infections in CF patients.

3′,5′-Cyclic Diguanylic Acid Reduces the Virulence of Biofilm-Forming Staphylococcus aureus Strains in a Mouse Model of Mastitis Infection

ABSTRACT The cyclic dinucleotide 3′,5′-cyclic diguanylic acid (c-di-GMP) is a naturally occurring small molecule that regulates important signaling systems in bacteria. We have recently shown that c-di-GMP inhibits Staphylococcus aureus biofilm formation in vitro and its adherence to HeLa cells. We now report that c-di-GMP treatment has an antimicrobial and antipathogenic activity in vivo and reduces, in a dose-dependent manner, bacterial colonization by biofilm-forming S. aureus strains in a mouse model of mastitis infection. Intramammary injections of 5 and 50 nmol of c-di-GMP decreased colonization (bacterial CFU per gram of gland) by 0.79 (P > 0.05) and 1.44 (P < 0.01) logs, respectively, whereas 200-nmol doses allowed clearance of the bacteria below the detection limit with a reduction of more than 4 logs (P < 0.001) compared to the untreated control groups. These results indicate that cyclic dinucleotides potentially represent an attractive and novel drug platform which could be used alone or in combination with other agents or drugs in the prevention, treatment, or control of infection.

c-di-GMP turn-over in Clostridium difficile is controlled by a plethora of diguanylate cyclases and phosphodiesterases.

Clostridium difficile infections have become a major healthcare concern in the last decade during which the emergence of new strains has underscored this bacteriums capacity to cause persistent epidemics. c-di-GMP is a bacterial second messenger regulating diverse bacterial phenotypes, notably motility and biofilm formation, in proteobacteria such as Vibrio cholerae, Pseudomonas aeruginosa, and Salmonella. c-di-GMP is synthesized by diguanylate cyclases (DGCs) that contain a conserved GGDEF domain. It is degraded by phosphodiesterases (PDEs) that contain either an EAL or an HD-GYP conserved domain. Very little is known about the role of c-di-GMP in the regulation of phenotypes of Gram-positive or fastidious bacteria. Herein, we exposed the main components of c-di-GMP signalling in 20 genomes of C. difficile, revealed their prevalence, and predicted their enzymatic activity. Ectopic expression of 31 of these conserved genes was carried out in V. cholerae to evaluate their effect on motility and biofilm formation, two well-characterized phenotype alterations associated with intracellular c-di-GMP variation in this bacterium. Most of the predicted DGCs and PDEs were found to be active in the V. cholerae model. Expression of truncated versions of CD0522, a protein with two GGDEF domains and one EAL domain, suggests that it can act alternatively as a DGC or a PDE. The activity of one purified DGC (CD1420) and one purified PDE (CD0757) was confirmed by in vitro enzymatic assays. GTP was shown to be important for the PDE activity of CD0757. Our results indicate that, in contrast to most Gram-positive bacteria including its closest relatives, C. difficile encodes a large assortment of functional DGCs and PDEs, revealing that c-di-GMP signalling is an important and well-conserved signal transduction system in this human pathogen.

Utilization of microbial iron assimilation processes for the development of new antibiotics and inspiration for the design of new anticancer agents

Pathogenic microbes rapidly develop resistance to antibiotics. To keep ahead in the “microbial war”, extensive interdisciplinary research is needed. A primary cause of drug resistance is the overuse of antibiotics that can result in alteration of microbial permeability, alteration of drug target binding sites, induction of enzymes that destroy antibiotics (ie., beta-lactamase) and even induction of efflux mechanisms. A combination of chemical syntheses, microbiological and biochemical studies demonstrate that the known critical dependence of iron assimilation by microbes for growth and virulence can be exploited for the development of new approaches to antibiotic therapy. Iron recognition and active transport relies on the biosyntheses and use of microbe-selective iron-chelating compounds called siderophores. Our studies, and those of others, demonstrate that siderophores and analogs can be used for iron transport-mediated drug delivery (“Trojan Horse” antibiotics) and induction of iron limitation/starvation (Development of new agents to block iron assimilation). Recent extensions of the use of siderophores for the development of novel potent and selective anticancer agents are also described.

The Fibronectin-Binding Proteins of Staphylococcus aureus May Promote Mammary Gland Colonization in a Lactating Mouse Model of Mastitis

ABSTRACT The fibronectin-binding proteins (FnBPs) of Staphylococcus aureus are believed to be implicated in the pathogens adherence to and colonization of bovine mammary glands, thus leading to infectious mastitis. In vitro studies have shown that FnBPs help the adhesion of the pathogen to bovine mammary epithelial cells. However, the importance of FnBPs for the infection of mammary glands has never been directly established in vivo. In this study with a mouse model of mastitis, the presence of FnBPs on the surface of S. aureus increased the capacity of the bacterium to colonize mammary glands under suckling pressure compared to that of a mutant lacking FnBPs.