Helicobacter pylori mutants defective in the clpP ATP-dependant protease and the chaperone clpA display reduced macrophage and murine survival.

The ATP-dependent caseinolytic proteases (Clp) are important in resistance against environmental stresses, antibiotic treatments and host immune defences for a number of pathogenic bacteria. ClpP is the proteolytic subunit, whilst ClpA acts both as a chaperone and as an ATPase driving the degradation of damaged or mis-made proteins. The gastric pathogen Helicobacter pylori infects approximately half of the world's population and can cause gastric or duodenal ulcers, gastric malignancies and mucosa-associated lymphoid tissue lymphomas. The conditions of its in vivo environment expose the organism to host immune cells and upon treatment, antibiotics, conditions likely to cause protein damage. We generated isogenic nonpolar mutants in strain SS1 of clpP and clpA and double mutants with both genes inactivated. Such mutants showed increased sensitivity to antibacterials causing protein damage and/or oxidative stress, in addition to a reduced survival in human macrophages. In the mouse infection model the double mutant SS1 clpAP lacked all ability to colonize the murine host. This suggests that the ability to recover from protein damage is of key importance in the pathogenesis of this organism.

Listeria monocytogenes biofilm-associated protein (BapL) may contribute to surface attachment of L. monocytogenes but is absent from many field isolates.

Listeria monocytogenes is a food-borne pathogen capable of adhering to a range of surfaces utilized within the food industry, including stainless steel. The factors required for the attachment of this ubiquitous organism to abiotic surfaces are still relatively unknown. In silico analysis of the L. monocytogenes EGD genome identified a putative cell wall-anchored protein (Lmo0435 [BapL]), which had similarity to proteins involved in biofilm formation by staphylococci. An insertion mutation was constructed in L. monocytogenes to determine the influence of this protein on attachment to abiotic surfaces. The results show that the protein may contribute to the surface adherence of strains that possess BapL, but it is not an essential requirement for all L. monocytogenes strains. Several BapL-negative field isolates demonstrated an ability to adhere to abiotic surfaces equivalent to that of BapL-positive strains. BapL is not required for the virulence of L. monocytogenes in mice.

Suicide through stress: a bacterial response to sub-lethal injury in the food environment.

The response of bacteria to sub-lethal injury is an important aspect of food microbiology as many inimical processes to which bacteria are subjected during processing are non-lethal. For pathogens like Salmonella and Escherichia coli, the difference in injury levels of exponential phase cells compared to their stationary phase counterparts in this regard is well recognised and evident for a variety of inimical processes. The expression of a range of stress resistance genes under the control of the sigma factor RpoS provides some explanation for the greater resistance of stationary phase cells. However in 1997 the suicide response hypothesis was put forward as an explanation for the observed response of Salmonella and E. coli to sub-lethal stresses. This hypothesis arose as an explanation for the observed protection of Salmonella and E. coli strains to heat and freeze-thaw injury by the presence of a high level of competitor organisms, a protection that had been shown to be RpoS independent. The central tenet of this theory was that under sub-lethal stress bacteria produce a burst of intracellular free radicals and it is these that lead to sub-lethal injury and/or death. Exponential phase cells because of their more active metabolism are more susceptible to this effect and suffer greater damage. This paper reviews the origins of this theory, the evidence for a free radical response and explores the potential mechanisms by which competitor cells produce a protective effect.

The resistance to detachment of dairy strains of Listeria monocytogenes from stainless steel by shear stress is related to the fluid dynamic characteristics of the location of isolation.

Strains of Listeria monocytogenes isolated from artisanal Portuguese cheese-making dairies were divided into two categories on the basis of the locations from which they were isolated: strains from dynamic locations were those that were habitually exposed to flowing liquids during the process of cheese-making, whereas those from static locations were rarely, if ever, exposed to the shear stresses generated by liquid flows. The strength of attachment to stainless steel discs of all of these strains was obtained using a radial flow chamber. Initial attachment strengths to stainless steel (after a 0.5 h contact time) of L. monocytogenes strains were greater for the 5 isolates from surfaces exposed to flow (dynamic isolates) than for most (3 out of 4) of those that were not (static isolates). After a 24 h contact time, attachment strength of all isolates reached similar levels. These results suggest that strains having high initial attachment strength are more likely to persist on surfaces exposed to flow than strains having low initial attachment strength. The numerical values of shear forces obtained could prove useful in the rational design of cleaning and decontamination procedures in food processing facilities.

The importance of RpoS in the survival of bacteria through food processing.

The resistance of bacteria to environmental stresses is recognised as an increasingly important area of microbiology. In particular, the alternative sigma factor RpoS has been shown to produce greater stress resistance in stationary phase cells of Salmonella and Escherichia coli compared with those in exponential phase. Our work has shown that RpoS can be induced in exponential phase in response to a number of inimical processes used in the food industry, including changes in water activity produced using a range of humectants and preservatives. The presence of high levels of competitor cells will also lead to early induction of RpoS in Salmonella by an as yet unknown mechanism. High levels of competitor cells also provide Salmonella with an increased resistance to heat and freeze-thaw injury; the mechanism for this, however, is rpoS independent and has lead to the theory of a holistic mechanism for sub-lethal injury in respiring bacteria--the bacterial suicide response. This hypothesis predicts that sub-lethal injury occurs through the production of free radical species and not by the action of the applied inimical process per se. The demonstration of the production of a free radical burst when cells are subjected to differing types of stresses has been shown by a number of methods.