Weng C. Chan

Look who's talking: communication and quorum sensing in the bacterial world

For many years bacteria were considered primarily as autonomous unicellular organisms with little capacity for collective behaviour. However, we now appreciate that bacterial cells are in fact, highly communicative. The generic term ‘quorum sensing’ has been adopted to describe the bacterial cell-to-cell communication mechanisms which co-ordinate gene expression usually, but not always, when the population has reached a high cell density. Quorum sensing depends on the synthesis of small molecules (often referred to as pheromones or autoinducers) that diffuse in and out of bacterial cells. As the bacterial population density increases, so does the synthesis of quorum sensing signal molecules, and consequently, their concentration in the external environment rises. Once a critical threshold concentration has been reached, a target sensor kinase or response regulator is activated (or repressed) so facilitating the expression of quorum sensing-dependent genes. Quorum sensing enables a bacterial population to mount a co-operative response that improves access to nutrients or specific environmental niches, promotes collective defence against other competitor prokaryotes or eukaryotic defence mechanisms and facilitates survival through differentiation into morphological forms better able to combat environmental threats. Quorum sensing also crosses the prokaryotic–eukaryotic boundary since quorum sensing-dependent signalling can be exploited or inactivated by both plants and mammals.

Human defensin 5 is stored in precursor form in normal Paneth cells and is expressed by some villous epithelial cells and by metaplastic Paneth cells in the colon in inflammatory bowel disease

BACKGROUND AND AIMS Intestinal epithelial cell derived antimicrobial peptides of the defensin family may play a major role in host defence against microorganisms. Our aims were to (i) isolate, characterise, and investigate the processing of human defensin 5 (HD-5) in normal Paneth cells and (ii) investigate expression of HD-5 in active inflammatory bowel disease (IBD). METHODS Antiserum raised against chemically synthesised putative mature HD-5 was used for immunohistochemistry and purification of HD-5 from extracts of normal terminal ileal crypts. RESULTS In normal and Crohns disease terminal ileum, HD-5 immunoreactivity was seen in Paneth cells and in some villous epithelial cells. Normal colonic mucosa did not express HD-5 but HD-5 immunoreactivity was seen in cells in the colonic crypt region of many IBD samples.N-terminal amino acid sequence analysis of HD-5 purified from normal terminal ileal Paneth cells consistently showed the predicted sequence of the precursor form of the peptide. Following stimulation of isolated intact normal terminal ileal crypts, a truncated form of HD-5, with theN-terminal sequence GEDNQLAIS, was detected in the supernatant. CONCLUSIONS (i) HD-5 is present only in the precursor form in normal terminal ileal Paneth cells and is processed to the mature form during and/or after secretion, (ii) some villous epithelial cells express HD-5, and (iii) HD-5 is expressed by metaplastic Paneth cells in the colon in IBD.

Poly(l-lysine)–GRGDS as a biomimetic surface modifier for poly(lactic acid)

The immobilization of adhesion peptide sequences (such as RGD) at the surfaces of poly(alpha-hydroxyacid)s, including poly(lactic acid) (PLA), is complicated by an absence of functional groups to support covalent attachment. We demonstrate a method to overcome this problem, by attaching the peptide to poly(L-lysine) (PLL), which immobilizes the sequence through adsorption at the poly(alpha-hydroxyacid) surface. When coated using a 0.01% w/v solution of PLL-GRGDS, bovine aortic endothelial cells seeded upon the modified PLA showed a marked increase in spreading over unmodified PLA. However, inhibition of the cell-spreading effect occurred when using higher concentrations of PLL-GRGDS, which we attribute to the PLL component. This inhibitory effect can be challenged by increasing the amount of GRGDS attached to each PLL molecule. Potentially, this is a flexible method of surface modification that can engineer many different types of tissue engineering scaffolds with a variety of biomolecules, thus allowing initial cell adhesion to be controlled.

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.

An appraisal of new variants of Dde amine protecting group for solid phase peptide synthesis

Abstract A series of new variants of Dde, substituted at the exocyclic alkene position have been developed and their application in solid phase peptide synthesis assessed. The new derivatives offer complete resistance towards 20% piperidine in DMF and provide a considerable barrier towards N→N′-intramolecular migration.

Structure‐activity relationships in the peptide antibiotic nisin: antibacterial activity of fragments of nisin

The post‐translationally modified peptide antibiotic nisin has been cleaved by a number of proteases and the fragments produced purified, characterised chemically, and assayed for activity in inhibiting the growth of Lactococcus lactis MG1614 and Micrococcus luteus NCDO8166. These results provide information on the importance of different parts of the nisin molecule for its growth‐inhibition activity. Removal of the C‐terminal five residues leads to approximately a 10‐fold decrease in potency, while removal of a further nine residues, encompassing two of the lanthionine rings, leads to a 100‐fold decrease. There are some differences between analogous fragments of nisin and subtilin, suggesting possible subtle differences in mode of action. Cleavage within, or removal of, lanthionine ring C essentially abolishes the activity of nisin. The fragment nisin1−12 is inactive itself, and specifically antagonises the growth‐inhibitory action of nisin. These results are discussed in terms of current models for the mechanism of action of nisin.

Isolation and characterisation of two degradation products derived from the peptide antibiotic nisin

Two degradation products of nisin have been isolated and their structures have been determined by 1H NMR. Nisin1–32 [(des‐ΔAla33‐Lys34; Val32‐NH2)nisin] and (des‐ΔAla5)nisin1–32 [(des‐ΔAla5, ΔAla33‐Lys34; Ile4‐NH2, pyruvyl‐Leu6, Val32‐NH2)nisin] are formed on storage or by acid treatment. Contrary to previous reports, nisin1–32 showed potent antimicrobial activity against Gram‐positive organisms comparable to that of nisin itself. (des‐ΔAla5)Nisin1–32, however, was biologically inactive, thus demonstrating the importance of ΔAla5 and/or ring A for biological activity.

N-Fmoc-aminooxy-2-chlorotrityl polystyrene resin: A facile solid-phase methodology for the synthesis of hydroxamic acids☆

Abstract Using the new compound N -Fmoc-hydroxylamine, 1 we have generated a facile route to a high loading, acid labile solid-phase resin bearing a hydroxylamine linker. The novel N -Fmoc-aminooxy-2-chlorotrityl polystyrene 2 showed generic utility for the construction of hydroxamic acids, including peptidyl hydroxamic acids.

Methicillin Resistance Reduces the Virulence of Healthcare-Associated Methicillin-Resistant Staphylococcus aureus by Interfering With the agr Quorum Sensing System

The difficulty in successfully treating infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has led to them being referred to as highly virulent or pathogenic. In our study of one of the major healthcare-associated MRSA (HA-MRSA) clones, we show that expression of the gene responsible for conferring methicillin resistance (mecA) is also directly responsible for reducing the ability of HA-MRSA to secrete cytolytic toxins. We show that resistance to methicillin induces changes in the cell wall, which affects the bacterias agr quorum sensing system. This leads to reduced toxin expression and, as a consequence, reduced virulence in a murine model of sepsis. This diminished capacity to cause infection may explain the inability of HA-MRSA to move into the community and help us understand the recent emergence of community-associated MRSA (CA-MRSA). CA-MRSA typically express less penicillin-binding protein 2a (encoded by mecA), allowing them to maintain full virulence and succeed in the community environment.