Alan Cockayne

The role of toxin A and toxin B in Clostridium difficile infection

Clostridium difficile infection is the leading cause of healthcare-associated diarrhoea in Europe and North America. During infection, C. difficile produces two key virulence determinants, toxin A and toxin B. Experiments with purified toxins have indicated that toxin A alone is able to evoke the symptoms of C. difficile infection, but toxin B is unable to do so unless it is mixed with toxin A or there is prior damage to the gut mucosa. However, a recent study indicated that toxin B is essential for C. difficile virulence and that a strain producing toxin A alone was avirulent. This creates a paradox over the individual importance of toxin A and toxin B. Here we show that isogenic mutants of C. difficile producing either toxin A or toxin B alone can cause fulminant disease in the hamster model of infection. By using a gene knockout system to inactivate the toxin genes permanently, we found that C. difficile producing either one or both toxins showed cytotoxic activity in vitro that translated directly into virulence in vivo. Furthermore, by constructing the first ever double-mutant strain of C. difficile, in which both toxin genes were inactivated, we were able to completely attenuate virulence. Our findings re-establish the importance of both toxin A and toxin B and highlight the need to continue to consider both toxins in the development of diagnostic tests and effective countermeasures against C. difficile.

agr Expression Precedes Escape of Internalized Staphylococcus aureus from the Host Endosome

ABSTRACT Staphylococcus aureus is a versatile pathogen capable of causing life-threatening infections. Many of its cell wall and exoproduct virulence determinants are controlled via the accessory gene regulator (agr). Although considered primarily as an extracellular pathogen, it is now recognized that S. aureus can be internalized by epithelial and endothelial cells. Traditional experimental approaches to investigate bacterial internalization are extremely time-consuming and notoriously irreproducible. We present here a new reporter gene method to assess intracellular growth of S. aureus in MAC-T cells that utilizes a gfp-luxABCDE reporter operon under the control of the Bacillus megateriumxylA promoter, which in S. aureus is expressed in a growth-dependent manner. This facilitates assessment of the growth of internalized bacteria in a nondestructive assay. The dual gfp-lux reporter cassette was also evaluated as a reporter of agr expression and used to monitor the temporal induction of agr during the MAC-T internalization process. The data obtained suggest thatagr induction occurs prior to endosomal lysis and thatagr-regulated exoproteins appear to be required prior to the release and replication of S. aureus within the infected MAC-T cells.

Structure, activity and evolution of the group I thiolactone peptide quorum‐sensing system of Staphylococcus aureus

In Staphylococcus aureus, the agr locus is responsible for controlling virulence gene expression via quorum sensing. As the blockade of quorum sensing offers a novel strategy for attenuating infection, we sought to gain novel insights into the structure, activity and turnover of the secreted staphylococcal autoinducing peptide (AIP) signal molecules. A series of analogues (including the l‐alanine and d‐amino acid scanned peptides) was synthesized to determine the functionally critical residues within the S. aureus group I AIP. As a consequence, we established that (i) the group I AIP is inactivated in culture supernatants by the formation of the corresponding methionyl sulphoxide; and (ii) the group I AIP lactam analogue retains the capacity to activate agr, suggesting that covalent modification of the AgrC receptor is not a necessary prerequisite for agr activation. Although each of the d‐amino acid scanned AIP analogues retained activity, replacement of the endocyclic amino acid residue (aspartate) located C‐terminally to the central cysteine with alanine converted the group I AIP from an activator to a potent inhibitor. The screening of clinical S. aureus isolates for novel AIP groups revealed a variant that differed from the group I AIP by a single amino acid residue (aspartate to tyrosine) in the same position defined as critical by alanine scanning. Although this AIP inhibits group I S. aureus strains, the producer strains possess a functional agr locus dependent on the endogenous peptide and, as such, constitute a fourth S. aureus AIP pheromone group (group IV). The addition of exogenous synthetic AIPs to S. aureus inhibited the production of toxic shock syndrome toxin (TSST‐1) and enterotoxin C3, confirming the potential of quorum‐sensing blockade as a therapeutic strategy.

N-Acylhomoserine Lactones Antagonize Virulence Gene Expression and Quorum Sensing in Staphylococcus aureus

ABSTRACT Many gram-negative bacteria employ N-acylhomoserine lactone (AHL)-mediated quorum sensing to control virulence. To determine whether gram-positive bacteria such as Staphylococcus aureus respond to AHLs, we used a growth-dependent lux reporter fusion. Exposure of S. aureus to different AHLs revealed that 3-oxo-substituted AHLs with C10 to C14 acyl chains inhibited light output and growth in a concentration-dependent manner, while short-chain AHLs had no effect. N-(3-Oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) inhibited the production of exotoxins and cell wall fibronectin-binding proteins but enhanced protein A expression. Since these processes are reciprocally regulated via the S. aureus agr quorum-sensing system, which in turn, is regulated via sar, we examined the effect of AHLs on sarA and agr. At sub-growth-inhibitory concentrations of 3-oxo-C12-HSL, both sarA expression and agr expression were inhibited, indicating that the action of 3-oxo-C12-HSL is mediated at least in part through antagonism of quorum sensing in S. aureus. Spent culture supernatants from Pseudomonas aeruginosa, which produces both 3-oxo-C12-HSL and N-butanoyl-homoserine lactone (C4-HSL), also inhibited agr expression, although C4-HSL itself was inactive in this assay. Since quorum sensing in S. aureus depends on the activities of membrane-associated proteins, such as AgrB, AgrC, and AgrD, we investigated whether AHLs perturbed S. aureus membrane functionality by determining their influence on the membrane dipole potential. From the binding curves obtained, a dissociation constant of 7 μM was obtained for 3-oxo-C12-HSL, indicating the presence of a specific saturable receptor, whereas no binding was observed for C4-HSL. These data demonstrate that long-chain 3-oxo-substituted AHLs, such as 3-oxo-C12-HSL, are capable of interacting with the S. aureus cytoplasmic membrane in a saturable, specific manner and at sub-growth-inhibitory concentrations, down-regulating exotoxin production and both sarA and agr expression.

Crystal structure of the heme-IsdC complex, the central conduit of the Isd iron/heme uptake system in staphylococcus aureus

Pathogens such as Staphylococcus aureus require iron to survive and have evolved specialized proteins to steal heme from their host. IsdC is the central conduit of the Isd (iron-regulated surface determinant) multicomponent heme uptake machinery; staphylococcal cell-surface proteins such as IsdA, IsdB, and IsdH are thought to funnel their molecular cargo to IsdC, which then mediates the transfer of the iron-containing nutrient to the membrane translocation system IsdDEF. The structure of the heme-IsdC complex reveals a novel heme site within an immunoglobulin-like domain and sheds light on its binding mechanism. The folding topology is reminiscent of the architecture of cytochrome f, cellobiose dehydrogenase, and ethylbenzene dehydrogenase; in these three proteins, the heme is bound in an equivalent position, but interestingly, IsdC features a distinct binding pocket with the ligand located next to the hydrophobic core of the β-sandwich. The iron is coordinated with a tyrosine surrounded by several non-polar side chains that cluster into a tightly packed proximal side. On the other hand, the distal side is relatively exposed with a short helical peptide segment that acts as a lip clasping onto almost half of the porphyrin plane. This structural feature is argued to play a role in the mechanism of binding and release by switching to an open conformation and thus loosening the interactions holding the heme. The structure of the heme-IsdC complex provides a template for the understanding of other proteins, such as IsdA, IsdB, and IsdH, that contain the same heme-binding module as IsdC, known as the NEAT (near transporter) domain.

Importance of Toxin A, Toxin B, and CDT in Virulence of an Epidemic Clostridium difficile Strain

Clostridium difficile infection is the main cause of healthcare-acquired diarrhea in the developed world. In addition to the main virulence factors toxin A and B, epidemic, PCR Ribotype 027 strains, such as R20291, produce a third toxin, CDT. To develop effective medical countermeasures, it is important to understand the importance of each toxin. Accordingly, we created all possible combinations of isogenic toxin mutants of R20291 and assessed their virulence. We demonstrated that either toxin A or toxin B alone can cause fulminant disease in the hamster infection model and present tantalizing data that C. difficile toxin may also contribute to virulence.

Synthesis and Deformylation of Staphylococcus aureus δ-Toxin Are Linked to Tricarboxylic Acid Cycle Activity

In bacteria, translation initiates with formyl-methionine; however, the N-terminal formyl group is usually removed by peptide deformylase, an enzymatic activity requiring iron. Staphylococcus aureus delta-toxin is a 26-amino-acid polypeptide secreted predominantly with a formylated N-terminal methionine, which led us to investigate regulation of delta-toxin deformylation. We observed that during exponential and early postexponential growth, delta-toxin accumulated in the culture medium in formylated and deformylated forms. In contrast, only formylated delta-toxin accumulated after the early postexponential phase. The transition from producing both species of delta-toxin to producing only formyl-methionine-containing delta-toxin coincided with increased tricarboxylic acid (TCA) cycle activity. The TCA cycle contains several iron-requiring enzymes, which led us to hypothesize that TCA cycle induction depletes the iron in the culture medium, thereby inhibiting peptide deformylase activity. As expected, S. aureus depletes the iron in the culture medium between the postexponential and stationary phases of growth. Inhibition of delta-toxin deformylation was relieved by TCA cycle inactivation or by addition of supplemental iron to the culture medium. Of interest, peptides containing formyl-methionine are potent chemoattractants for neutrophils, suggesting that delta-toxin deformylation may have functional consequences. We found neutrophil chemotactic activity only with formylated delta-toxin. The S. aureus TCA cycle is derepressed upon depletion of rapidly catabolizable carbon sources; this coincides with the transition to producing only formylated delta-toxin and results in an increased inflammatory response. The proinflammatory response should increase host cell damage and result in the release of nutrients. Taken together, these results establish that there is an important linkage between bacterial metabolism and pathogenesis.

Iron-Regulated Biofilm Formation in Staphylococcus aureus Newman Requires ica and the Secreted Protein Emp

ABSTRACT Staphylococcus aureus biofilm formation is induced in iron-restricted growth conditions in vitro. In this study, we showed that Emp and Eap play important roles in low-iron-induced biofilm formation of S. aureus Newman. Eap and Emp are secreted proteins which are non-covalently attached to the S. aureus cell surface and have previously been implicated in a number of aspects of S. aureus pathogenesis. We showed here that the transcription of these important virulence factors is induced by growth in low-iron medium, reflective of the in vivo environment. Our results show that iron regulation of Eap and Emp is Fur independent. However, Fur is required for full induction of eap and emp expression in low-iron conditions. In this study, we demonstrated that in addition to Fur, low-iron-induced biofilm formation requires Sae, Agr, and SarA. In iron-restricted growth conditions, Sae and Agr are essential for Emp and Eap expression and hence for biofilm formation, whereas SarA appears to have a less-significant role. We also showed that expression of the ica operon is required for biofilm formation in iron-restricted growth conditions. We demonstrated that in fact, ica is required for the expression of the important multifunctional virulence determinants eap and emp.

Stable expression of foreign antigens from the chromosome of Salmonella typhimurium vaccine strains.

A simple and versatile system has been developed using a new cloning vector which can serve as a vehicle for integrating DNA fragments, which direct the expression of heterologous antigens, into the aroC gene on the Salmonella chromosome. The system is based on Escherichia coli plasmid vectors which contain the DNA fragment, cloned from the chromosome of S. typhimurium C5, which encodes the aroC gene. The aroC gene was modified using synthetic oligodeoxyribonucleotides so that it contained several unique restriction sites into which DNA, directing the expression of heterologous antigens, could be cloned. DNA was integrated into the S. typhimurium chromosome at aroC by transferring the vectors into S. typhimurium polA mutants and allowing homologous recombination to occur between the cloned and chromosomal aroC genes. The vectors were used to integrate nucleotide sequences into the S. typhimurium chromosome which directed the expression of tetanus toxin fragment C and the Treponema pallidum lipoprotein. The expression of both antigens was detected by Western blotting.

Iron-Responsive Regulation of Biofilm Formation in Staphylococcus aureus Involves Fur-Dependent and Fur-Independent Mechanisms

We have shown that Staphylococcus aureus biofilm production is induced in iron-restricted conditions and is repressed by iron via a Fur-independent mechanism, while Fur has both positive and negative regulatory roles in low iron. Furthermore, there is no significant increase in polymeric N-acetylglucosamine polysaccharide expression to account for induction of biofilms in low iron.