Kristoffer T. Bæk

Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin

BackgroundCampylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear.ResultsIn the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni’s HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori’s HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria.ConclusionThese results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

Antibiotic-Mediated Selection of Quorum-Sensing-Negative Staphylococcus aureus

ABSTRACT Staphylococcus aureus is a human commensal that at times turns into a serious bacterial pathogen causing life-threatening infections. For the delicate control of virulence, S. aureus employs the agr quorum-sensing system that, via the intracellular effector molecule RNAIII, regulates virulence gene expression. We demonstrate that the presence of the agr locus imposes a fitness cost on S. aureus that is mediated by the expression of RNAIII. Further, we show that exposure to sublethal levels of the antibiotics ciprofloxacin, mupirocin, and rifampin, each targeting separate cellular functions, markedly increases the agr-mediated fitness cost by inducing the expression of RNAIII. Thus, the extensive use of antibiotics in hospitals may explain why agr-negative variants are frequently isolated from hospital-acquired S. aureus infections but rarely found among community-acquired S. aureus strains. Importantly, agr deficiency correlates with increased duration of and mortality due to bacteremia during antibiotic treatment and with a higher frequency of glycopeptide resistance than in agr-carrying strains. Our results provide an explanation for the frequent isolation of agr-defective strains from hospital-acquired S. aureus infections and suggest that the adaptability of S. aureus to antibiotics involves the agr locus. IMPORTANCE Staphylococcus aureus is the most frequently isolated pathogen in intensive care units and a common cause of nosocomial infections, resulting in a high degree of morbidity and mortality. Surprisingly, a large fraction (15 to 60%) of hospital-isolated S. aureus strains are agr defective and lack the main quorum-sensing-controlled virulence regulatory system. This is a problem, as agr-defective strains are associated with a mortality level in bacteremic infections and a probability of glycopeptide resistance greater than those of other strains. We show here that agr-negative strains have a fitness advantage over agr-positive strains in the presence of sublethal concentrations of some antibiotics and that the fitness defect of agr-positive cells is caused by antibiotic-mediated expression of the agr effector molecule RNAIII. These results offer an explanation of the frequent isolation of agr-defective S. aureus strains in hospitals and will influence how we treat S. aureus infections. Staphylococcus aureus is the most frequently isolated pathogen in intensive care units and a common cause of nosocomial infections, resulting in a high degree of morbidity and mortality. Surprisingly, a large fraction (15 to 60%) of hospital-isolated S. aureus strains are agr defective and lack the main quorum-sensing-controlled virulence regulatory system. This is a problem, as agr-defective strains are associated with a mortality level in bacteremic infections and a probability of glycopeptide resistance greater than those of other strains. We show here that agr-negative strains have a fitness advantage over agr-positive strains in the presence of sublethal concentrations of some antibiotics and that the fitness defect of agr-positive cells is caused by antibiotic-mediated expression of the agr effector molecule RNAIII. These results offer an explanation of the frequent isolation of agr-defective S. aureus strains in hospitals and will influence how we treat S. aureus infections.

New insights into Staphylococcus aureus stress tolerance and virulence regulation from an analysis of the role of the ClpP protease in the strains Newman, COL, and SA564.

In Staphylococcus aureus, ClpP proteases were previously shown to be essential for virulence and stress tolerance in strains derived from NCTC8325. Because these strains exhibit a severely reduced activity of the alternative sigma factor, SigB, we here reassessed the role of ClpP in SigB-proficient clinical strains. To this end, clpP was deleted in strains COL, Newman, and SA564, and the strains were characterized phenotypically. The proteomic changes accomplished by the clpP deletion in the different strains were analyzed using the 2-D DIGE technique. The proteomic analyses revealed mostly conserved changes in the protein profiles of the ClpP-deficient strains. Among the strain-specific changes were the up-regulation of prophage proteins that coincided with an increased spontaneous release of prophages and the relatively poorer growth of the clpP mutants in some strain backgrounds. Interestingly, the effect of ClpP on the expression of selected virulence genes was strain-dependent despite the fact that the expression of the global virulence regulators RNAIII, mgrA, sarZ, sarR, and arlRS was similarly changed in all clpP mutants. ClpP affected the expression of sarS in a strain-dependent manner, and we propose that the differential expression of sarS is central to the strain-dependent effect of ClpP on the expression of virulence genes.

HtrA chaperone activity contributes to host cell binding in Campylobacter jejuni

BackgroundAcute gastroenteritis caused by the food-borne pathogen Campylobacter jejuni is associated with attachment of bacteria to the intestinal epithelium and subsequent invasion of epithelial cells. In C. jejuni, the periplasmic protein HtrA is required for efficient binding to epithelial cells. HtrA has both protease and chaperone activity, and is important for virulence of several bacterial pathogens.ResultsThe aim of this study was to determine the role of the dual activities of HtrA in host cell interaction of C. jejuni by comparing an htrA mutant lacking protease activity, but retaining chaperone activity, with a ΔhtrA mutant and the wild type strain. Binding of C. jejuni to both epithelial cells and macrophages was facilitated mainly by HtrA chaperone activity that may be involved in folding of outer membrane adhesins. In contrast, HtrA protease activity played only a minor role in interaction with host cells.ConclusionWe show that HtrA protease and chaperone activities contribute differently to C. jejunis interaction with mammalian host cells, with the chaperone activity playing the major role in host cell binding.

Different Contributions of HtrA Protease and Chaperone Activities to Campylobacter jejuni Stress Tolerance and Physiology

ABSTRACT The microaerophilic bacterium Campylobacter jejuni is the most common cause of bacterial food-borne infections in the developed world. Tolerance to environmental stress relies on proteases and chaperones in the cell envelope, such as HtrA and SurA. HtrA displays both chaperone and protease activities, but little is known about how each of these activities contributes to stress tolerance in bacteria. In vitro experiments showed temperature-dependent protease and chaperone activities of C. jejuni HtrA. A C. jejuni mutant lacking only the protease activity of HtrA was used to show that the HtrA chaperone activity is sufficient for growth at high temperature or under oxidative stress, whereas the HtrA protease activity is essential only under conditions close to the growth limit for C. jejuni. However, the protease activity was required to prevent induction of the cytoplasmic heat shock response even under optimal growth conditions. Interestingly, the requirement of HtrA at high temperatures was found to depend on the oxygen level, and our data suggest that HtrA may protect oxidatively damaged proteins. Finally, protease activity stimulates HtrA production and oligomer formation, suggesting that a regulatory role depends on the protease activity of HtrA. Studying a microaerophilic organism encoding only two known periplasmic chaperones (HtrA and SurA) revealed an efficient HtrA chaperone activity and proposed multiple roles of the protease activity, increasing our understanding of HtrA in bacterial physiology.

Genetic Variation in the Staphylococcus aureus 8325 Strain Lineage Revealed by Whole-Genome Sequencing

Staphylococcus aureus strains of the 8325 lineage, especially 8325-4 and derivatives lacking prophage, have been used extensively for decades of research. We report herein the results of our deep sequence analysis of strain 8325-4. Assignment of sequence variants compared with the reference strain 8325 (NRS77/PS47) required correction of errors in the 8325 reference genome, and reassessment of variation previously attributed to chemical mutagenesis of the restriction-defective RN4220. Using an extensive strain pedigree analysis, we discovered that 8325-4 contains 16 single nucleotide polymorphisms (SNP) arising prior to the construction of RN4220. We identified 5 indels in 8325-4 compared with 8325. Three indels correspond to expected Φ11, 12, 13 excisions, one indel is explained by a sequence assembly artifact, and the final indel (Δ63bp) in the spa-sarS intergenic region is common to only a sub-lineage of 8325-4 strains including SH1000. This deletion was found to significantly decrease (75%) steady state sarS but not spa transcript levels in post-exponential phase. The sub-lineage 8325-4 was also found to harbor 4 additional SNPs. We also found large sequence variation between 8325, 8325-4 and RN4220 in a cluster of repetitive hypothetical proteins (SA0282 homologs) near the Ess secretion cluster. The overall 8325-4 SNP set results in 17 alterations within coding sequences. Remarkably, we discovered that all tested strains of the 8325-4 lineage lack phenol soluble modulin α3 (PSMα3), a virulence determinant implicated in neutrophil chemotaxis, biofilm architecture and surface spreading. Collectively, our results clarify and define the 8325-4 pedigree and reveal clear evidence that mutations existing throughout all branches of this lineage, including the widely used RN6390 and SH1000 strains, could conceivably impact virulence regulation.

β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus USA300 Is Increased by Inactivation of the ClpXP Protease

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) has acquired the mecA gene encoding a peptidoglycan transpeptidase, penicillin binding protein 2a (PBP2a), which has decreased affinity for β-lactams. Quickly spreading and highly virulent community-acquired (CA) MRSA strains recently emerged as a frequent cause of infection in individuals without exposure to the health care system. In this study, we found that the inactivation of the components of the ClpXP protease substantially increased the β-lactam resistance level of a CA-MRSA USA300 strain, suggesting that the proteolytic activity of ClpXP controls one or more pathways modulating β-lactam resistance. These pathways do not involve the control of mecA expression, as the cellular levels of PBP2a were unaltered in the clp mutants. An analysis of the cell envelope properties of the clpX and clpP mutants revealed a number of distinct phenotypes that may contribute to the enhanced β-lactam tolerance. Both mutants displayed significantly thicker cell walls, increased peptidoglycan cross-linking, and altered composition of monomeric muropeptide species compared to those of the wild types. Moreover, changes in Sle1-mediated peptidoglycan hydrolysis and altered processing of the major autolysin Atl were observed in the clp mutants. In conclusion, the results presented here point to an important role for the ClpXP protease in controlling cell wall metabolism and add novel insights into the molecular factors that determine strain-dependent β-lactam resistance.

Stepwise Decrease in Daptomycin Susceptibility in Clinical Staphylococcus aureus Isolates Associated with an Initial Mutation in rpoB and a Compensatory Inactivation of the clpX Gene

ABSTRACT Daptomycin is a lipopeptide antibiotic used clinically for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. The emergence of daptomycin-nonsusceptible S. aureus isolates during therapy is often associated with multiple genetic changes; however, the relative contributions of these changes to resistance and other phenotypic changes usually remain unclear. The present study was undertaken to investigate this issue using a genetically characterized series of four isogenic clinical MRSA strains derived from a patient with bacteremia before and during daptomycin treatment. The first strain obtained after daptomycin therapy carried a single-nucleotide polymorphism (SNP) in rpoB (RpoB A477D) that decreased susceptibility not only to daptomycin but also to vancomycin, β-lactams, and rifampin. Furthermore, the rpoB mutant exhibited pleiotropic phenotypes, including increased cell wall thickness, reduced expression of virulence traits, induced expression of the stress-associated transcriptional regulator Spx, and slow growth. A subsequently acquired loss-of-function mutation in clpX partly alleviated the growth defect conferred by the rpoB mutation without changing antibiotic susceptibility. The final isolate acquired three additional mutations, including an SNP in mprF (MprF S295L) known to confer daptomycin nonsusceptibility, and accordingly, this isolate was the only daptomycin-nonsusceptible strain of this series. Interestingly, in this isolate, the cell wall had regained the same thickness as that of the parental strain, while the level of transcription of the vraSR (cell wall stress regulator) was increased. In conclusion, this study illustrates how serial genetic changes selected in vivo contribute to daptomycin nonsusceptibility, growth fitness, and virulence traits.

The Cell Wall Polymer Lipoteichoic Acid Becomes Nonessential in Staphylococcus aureus Cells Lacking the ClpX Chaperone

ABSTRACT Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria and a promising target for the development of vaccines and antimicrobial compounds against Staphylococcus aureus. Here we demonstrate that mutations in the conditionally essential ltaS (LTA synthase) gene arise spontaneously in an S. aureus mutant lacking the ClpX chaperone. A wide variety of ltaS mutations were selected, and among these, a substantial portion resulted in premature stop codons and other changes predicted to abolish LtaS synthesis. Consistent with this assumption, the clpX ltaS double mutants did not produce LTA, and genetic analyses confirmed that LTA becomes nonessential in the absence of the ClpX chaperone. In fact, inactivation of ltaS alleviated the severe growth defect conferred by the clpX deletion. Microscopic analyses showed that the absence of ClpX partly alleviates the septum placement defects of an LTA-depleted strain, while other phenotypes typical of LTA-negative S. aureus mutants, including increased cell size and decreased autolytic activity, are retained. In conclusion, our results indicate that LTA has an essential role in septum placement that can be bypassed by inactivating the ClpX chaperone. IMPORTANCE Lipoteichoic acid is an essential component of the Staphylococcus aureus cell envelope and an attractive target for the development of vaccines and antimicrobials directed against antibiotic-resistant Gram-positive bacteria such as methicillin-resistant S. aureus and vancomycin-resistant enterococci. In this study, we showed that the lipoteichoic acid polymer is essential for growth of S. aureus only as long as the ClpX chaperone is present in the cell. Our results indicate that lipoteichoic acid and ClpX play opposite roles in a pathway that controls two key cell division processes in S. aureus, namely, septum formation and autolytic activity. The discovery of a novel functional connection in the genetic network that controls cell division in S. aureus may expand the repertoire of possible strategies to identify compounds or compound combinations that kill antibiotic-resistant S. aureus. Lipoteichoic acid is an essential component of the Staphylococcus aureus cell envelope and an attractive target for the development of vaccines and antimicrobials directed against antibiotic-resistant Gram-positive bacteria such as methicillin-resistant S. aureus and vancomycin-resistant enterococci. In this study, we showed that the lipoteichoic acid polymer is essential for growth of S. aureus only as long as the ClpX chaperone is present in the cell. Our results indicate that lipoteichoic acid and ClpX play opposite roles in a pathway that controls two key cell division processes in S. aureus, namely, septum formation and autolytic activity. The discovery of a novel functional connection in the genetic network that controls cell division in S. aureus may expand the repertoire of possible strategies to identify compounds or compound combinations that kill antibiotic-resistant S. aureus.

Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus

UNLABELLED Staphylococcus aureus is capable of causing a remarkable spectrum of disease, ranging from mild skin eruptions to life-threatening infections. The survival and pathogenic potential of S. aureus depend partly on its ability to sense and respond to changes in its environment. Spx is a thiol/oxidative stress sensor that interacts with the C-terminal domain of the RNA polymerase RpoA subunit, leading to changes in gene expression that help sustain viability under various conditions. Using genetic and deep-sequencing methods, we show that spx is essential in S. aureus and that a previously reported Δspx strain harbored suppressor mutations that allowed it to grow without spx One of these mutations is a single missense mutation in rpoB (a P-to-L change at position 519 encoded by rpoB [rpoB-P519L]) that conferred high-level resistance to rifampin. This mutation alone was found to be sufficient to bypass the requirement for spx The generation of rifampin resistance libraries led to the discovery of an additional rpoB mutation, R484H, which supported strains with the spx disruption. Other rifampin resistance mutations either failed to support the Δspx mutant or were recovered at unexpectedly low frequencies in genetic transduction experiments. The amino acid residues encoded by rpoB-P519L and -R484H map in close spatial proximity and comprise a highly conserved region of RpoB. We also discovered that multicopy expression of either trxA (encoding thioredoxin) or trxB (encoding thioredoxin reductase) supports strains with the deletion of spx Our results reveal intriguing properties, especially of RNA polymerase, that compensate for the loss of an essential gene that is a key mediator of diverse processes in S. aureus, including redox and thiol homeostasis, antibiotic resistance, growth, and metabolism. IMPORTANCE The survival and pathogenicity of S. aureus depend on complex genetic programs. An objective for combating this insidious organism entails dissecting genetic regulatory circuits and discovering promising new targets for therapeutic intervention. In this study, we discovered that Spx, an RNA polymerase-interacting stress regulator implicated in many stress responses in S. aureus, including responses to oxidative and cell wall antibiotics, is essential. We describe two mechanisms that suppress the lethality of spx disruption. One mechanism highlights how only certain rifampin resistance-encoding alleles of RpoB confer new properties on RNA polymerase, with important mechanistic implications. We describe additional stress conditions where the loss of spx is deleterious, thereby highlighting Spx as a multifaceted regulator and attractive drug discovery target.