agrExpression Precedes Escape of InternalizedStaphylococcus aureusfrom 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 megaterium xylA 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 gfplux 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 that agr induction occurs prior to endosomal lysis and that agr-regulated exoproteins appear to be required prior to the release and replication of S. aureus within the infected MAC-T cells.Staphylococcus aureus is the etiologic agent of numerous infections in humans and domesticated animals and has been implicated in a multitude of diseases, ranging from minor wound infections to more serious diseases, including endocarditis, osteomyelitis, and septic shock (reviewed by Projan and Novick [34]). The expression of many S. aureus virulence factors is under the control of the accessory gene regulator (agr) which, on entering post-exponential phase, downregulates the production of cell surface-associated proteins and upregulates the expression of secreted toxins and extracellular enzymes (28,33,38). The role of the agr regulon is supported by in vivo studies, which show that agr mutants are greatly attenuated in several animal models, including intramammary infections (13), arthritis in mice (1), and endocarditis in rabbits (7). The agr locus is a quorum-sensing-regulated system activated by autoinducing peptide pheromone (AIP) (21, 25). The agr locus consists of two divergent transcriptional units, RNAII and RNAIII, which are under the control of the P2 and P3 promoters, respectively (reviewed by Novick and Muir [30]). RNAII is a polycistronic mRNA that encodes the agrB and agrD genes required for the synthesis of the AIP and also the two component signal transduction proteins, AgrA and AgrC, which are responsible for sensing and responding to the AIP. RNAIII is the effector molecule in the agr regulon acting primarily at the level of gene transcription. Different S. aureus strains produce AIPs with distinct structures, and strains can be grouped on this basis since they will activate the agr response of strains within the same group and inhibit the agr response of strains from different groups by competitive inhibition (21,30). This inhibitory action of AIPs has identi...

Fibrinogen Depletion Attenuates Staphyloccocus aureus Infection by Preventing Density-Dependent Virulence Gene Up-Regulation

“…Host microenvironments could potentiate quorum sensing either by increasing the concentration of the autoinducer, as we showed with clumping, or by limiting diffusion of the autoinducer, as may occur within an intracellular vacuole (39). Thus, virulence as a quorum sensingregulated behavior may not represent exclusively the absolute bacterial number but, rather, the microenvironment in the host surrounding the bacteria.…”

“…While agr-deficient bacteria are less virulent than WT (1, 24), they can cause serious infection and death when given at increased inocula (39,32,40). Therefore, we measured both morbidity and mortality in mice infected with an appropriate challenge dose of agr-deficient bacteria and found that fibrinogen depletion affected neither morbidity (Fig.…”

Bacterial luciferase reporters: The Swiss army knife of molecular biology

“…To overcome these problems, translational signals optimized for Gram-positive bacteria have been introduced in front of luxA, luxC and luxE resulting in enhanced expression in Staphylococcus aureus. 47 The same construct has been successfully used for bioluminescent labelling of Listeria monocytogenes, 48,49 Streptococcus pneumoniae, 50 Bifidobacterium breve 51,52 and Mycobacterium tuberculosis and M. smegmatis. 53 A similar approach, however introducing Gram-positive ribosome binding sites in front of all genes of the luxABCDE operon has been applied by others.…”

“…61 A promoter fusion to an optimized P. luminescens lux operon was used to measure expression of a peptide sensing system of S. aureus in response to the signalling peptide in vitro and during phagocytosis and intracellular growth in macrophages. 47 The same system was used to quantify activities of various promoters of L. monocytogenes during growth in batch culture, infection of epithelial cells in vitro and in organs of infected mice ex vivo 48,49,[62][63][64] (Fig. 3).…”

Imaging of light emission from the expression of luciferases in living cells and organisms: a review

“…Recently a gfp-luxABCDE reporter construct, under the control of the XylA promoter, was used to monitor the expression and temporal induction of the quorum-sensing accessory gene regulator (agr) in S. aureus infecting bovine mammary epithelial MAC-T cells (133). The reporter gene expression was occasioned by the virulence factor-mediated escape of S. aureus from the host endosome and its ensuing intracytoplasmic growth.…”