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Listeria monocytogenes, the causative agent of listeriosis, is a virulent foodborne Gram-positive bacterial pathogen, with 20-30% mortality. It has a broad ability to transport iron, either in the form of ferric siderophores, or by extracting it from mammalian iron binding proteins. In this review we focus on the mechanisms of ferric siderophore and haem transport into the listerial cell. Despite the fact that it does not synthesize siderophores, L. monocytogenes transports ferric siderophores in the wild environment by the actions of cytoplasmic membrane ABC-transporter systems. The bacterium acquires haem, on the other hand, by two mechanisms. At low (nanomolar) concentrations, sortase B-dependent, peptidoglycan-anchored proteins scavenge the iron porphyrin in human or animal tissues, and transfer it to the underlying ABC-transporters in the cytoplasmic membrane for uptake. At concentrations at or above 50 nM, however, haem transport becomes sortase-independent, and occurs by direct interactions of the iron porphyrin with the same ABC-transporter complexes. The architecture of the Gram-positive cell envelope plays a fundamental role in these mechanisms, and the haem acquisition abilities of L. monocytogenes are an element of its ability to cause infectious disease. [ABSTRACT FROM AUTHOR]

We studied three Fur-regulated systems of Listeria monocytogenes: the srtB region, that encodes sortase-anchored proteins and a putative ABC transporter, and the fhu and hup operons, that produce putative ABC transporters for ferric hydroxamates and haemin (Hn)/haemoglobin (Hb) respectively. Deletion of lmo2185 in the srtB region reduced listerial [Fe]-Hn transport, and purified Lmo2185 bound [Fe]-Hn ( K = 12 nM), leading to its designation as a n/b inding rotein ( hbp2) . Purified Hbp2 also acted as a haemophore, capturing and supplying Hn from the environment. Nevertheless, Hbp2 only functioned in [Fe]-Hn transport at external concentrations less than 50 nM: at higher Hn levels its uptake occurred with equivalent affinity and rate without Hbp2. Similarly, deletion of sortase A had no effect on ferric siderophore or Hn/Hb transport at any concentration, and the srtA-independence of listerial Hn/Hb uptake distinguished it from comparable systems of Staphylococcus aureus. In the cytoplasmic membrane, the Hup transporter was specific for Hn: its lipoprotein (HupD) only showed high affinity for the iron porphyrin ( K = 26 nM). Conversely, the FhuD lipoprotein encoded by the fhu operon had broad specificity: it bound both ferric siderophores and Hn, with the highest affinity for ferrioxamine B ( K = 123 nM). Deletions of Hup permease components hupD, hupG or hupDGC reduced Hn/Hb uptake, and complementation of ΔhupC and ΔhupG by chromosomal integration of hupC and hupG alleles on pPL2 restored growth promotion by Hn/Hb. However, ΔhupDGC did not completely eliminate [Fe]-Hn transport, implying the existence of another cytoplasmic membrane Hn transporter. The overall K of Hn uptake by wild-type strain EGD-e was 1 nM, and it occurred at similar rates ( V = 23 pmol 10 cells min) to those of ferric siderophore transporters. In the ΔhupDGC strain uptake occurred at a threefold lower rate ( V = 7 pmol 10 cells min). The results show that at low (< 50 nM) levels of Hn, SrtB-dependent peptidoglycan-anchored proteins (e.g. Hbp2) bind the porphyrin, and HupDGC or another transporter completes its uptake into the cytoplasm. However, at higher concentrations Hn uptake is SrtB-independent: peptidoglycan-anchored binding proteins are dispensable because HupDGC directly absorbs and internalizes Hn. Finally, ΔhupDGC increased the LD of L. monocytogenes 100-fold in the mouse infection model, reiterating the importance of this system in listerial virulence. [ABSTRACT FROM AUTHOR]

Cholesterol-dependent cytolysins (CDCs) are produced by a large number of pathogenic Gram-positive bacteria. Most of these single-chain proteins are secreted in the extracellular medium. Among the species producing CDCs, only two species belonging to the genus Listeria ( Listeria monocytogenes and Listeria ivanovii) are able to multiply intracellularly and release their toxins in the phagosomal compartment of the infected host cell. This review provides an updated overview on the importance of listeriolysin O (LLO) in the pathogenicity of L. monocytogenes, focusing mainly on two aspects: (1) the structure–function relationship of LLO and (2) its role in intra- and extracellular signalling. We first examine the specific sequence determinants, or protein domains, that make this cytolysin so well adapted to the intracellular lifestyle of L. monocytogenes. The roles that LLO has in cellular signalling events in the context of relations to pathogenesis are also discussed. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a Gram-positive bacterium that causes severe opportunistic infections in humans and animals. We biochemically characterized, for the first time, the iron uptake processes of this facultative intracellular pathogen, and identified the genetic loci encoding two of its membrane iron transporters. Strain EGD-e used iron complexes of hydroxamates (ferrichrome and ferrichrome A, ferrioxamine B), catecholates (ferric enterobactin, ferric corynebactin) and eukaryotic binding proteins (transferrin, lactoferrin, ferritin, haemoglobin). Quantitative determinations showed 10–100-fold lower affinity for ferric siderophores ( Km ≈ 1–10 nM) than Gram-negative bacteria, and generally lower uptake rates. Vmax for [59Fe]-enterobactin (0.15 pMol per 109 cells per minute) was 400-fold lower than that of Escherichia coli. For [59Fe]-corynebactin, Vmax was also low (1.2 pMol per 109 cells per minute), but EGD-e transported [59Fe]-apoferrichrome similarly to E. coli ( Vmax = 24 pMol per 109 cells per minute). L. monocytogenes encodes potential Fur-regulated iron transporters at 2.031 Mb (the fur-fhu region), 2.184 Mb (the feo region), 2.27 Mb (the srtB region) and 2.499 Mb (designated hupDGC region). Chromosomal deletions in the fur-fhu and hupDGC regions diminished iron uptake from ferric hydroxamates and haemin/haemoglobin respectively. In the former locus, deletion of fhuD ( lmo1959) or fhuC ( lmo1960) strongly reduced [59Fe]-apoferrichrome uptake. Deletion of hupC ( lmo2429) eliminated the uptake of haemin and haemoglobin, and decreased the virulence of L. monocytogenes 50-fold in mice. Elimination of srtB region genes (Δ lmo2185, Δ lmo2186, Δ lmo2183), both sortase structural genes (Δ srtB, Δ srtA, Δ srtAB), fur and feoB did not impair iron transport. However, deletion of bacterioferritin (Δ fri, lmo943; 0.97 Mb) decreased growth and altered iron uptake: Vmax of [59Fe]-corynebactin transport tripled in this strain, whereas that of [59Fe]-apoferrichrome decreased 20-fold. [ABSTRACT FROM AUTHOR]

InListeria monocytogenesthe promoter region of thesvpA-srtBlocus contains a well-conserved Fur box. We characterized the iron-regulation of this locus: real-time polymerase chain reaction analyses and anti-SvpA immunoblots showed that, in response to iron deprivationsvpAtranscription and SvpA production markedly increased (80-fold and 10-fold respectively), when initiated by either the addition of the iron chelator 2,2′-bipyridyl to BHI media, or by growth in iron-restricted minimal media. Green fluorescent protein (GFP) reporter constructs also showed increased activity of thesvpA-srtBpromoter inEscherichia coli (37-fold) and inL. monocytogenes(two- to threefold) when the bacteria were grown in iron-deficient conditions. AΔ furmutant ofL. monocytogenesconstitutively synthesized SvpA, as well as GFP fused to thesvpA-srtBpromoter. Cellular fractionation data revealed that in iron-rich media wild-type SvpA was exclusively secreted to the culture supernatant. However, both theΔ furderivative and wild-typeL. monocytogenesgrown in iron-deficient media anchored a fraction of the SvpA proteins (∼5%) to peptidoglycan, and produced a lower-molecular weight, wholly secreted form of SvpA. Together these data establish that iron availability controls transcription of thesvpA-srtBlocus (through Fur-mediated regulation), and attachment of SvpA to the cell wall (through SrtB-mediated covalent linkage). SvpA bears homology to IsdC, a haemin-binding protein ofStaphylococcus aureus,and haemin bound to SvpA in solution. However, site-directed deletions of four structural genes and the promoter of thesvpA-srtBlocus did not impair haemin, haemoglobin or ferrichrome utilization in nutrition tests. We did not find strong evidence to support the notion that thesvpA-srtBlocus participates in haemin acquisition, as was reported for the homologousisdoperon ofS. aureus. Furthermore, thesvpA-srtBmutant strains showed no significant attenuation of virulence in an intravenous mouse model system, but we found that the mutations reduced the persistence ofL. monocytogenesin murine liver, spleen and intestines after oral administration. [ABSTRACT FROM AUTHOR]

Lamb of Escherichia coli K12, also called maltoporin, is an outer membrane protein, which specifically facilitates the diffusion of maltose and maltodextrin through the bacterial outer membrane. Each monomer is composed of an 18-stranded antiparallel β-barrel. In the present work, on the basis of the known X-ray structure of LamB, the effects of modifications of the β-barrel domain of maltoporin were studied in vivo and in vitro. We show that: (i) the substitution of the pair of strands β13-β14 of the E. coli maltoporin with the corresponding pair of strands from the functionally related maltoporin of Salmonella typhimurium yielded a protein active in vivo and in vitro; and (ii) the removal of one pair of β-strands (deletion β13-β14) from the E. coli maltoporin, or its replacement by a pair of strands from the general porin OmpF of E. coli, leads to recombinant proteins that lost in vivo maltoporin activities but still kept channel formation and carbohydrate binding in vitro. We also inserted into deletion β13-β14 the portion of the E. coli Lamb protein comprising strands β13 to β16. This resulted in a protein expected to have 20 β-strands and which completely lost all LamB-specific activities in vivo and in vitro. [ABSTRACT FROM AUTHOR]

The trimeric protein LamB of Escherichia coli K-12 (maltoporin) specifically facilitates the diffusion of maltose and maltooligosaccharides through the outer membrane. Each monomer consists of an 18-stranded antiparallel β-barrel with nine surface loops (L1 to L9). The effects on transport and binding of the deletion of some of the surface loops or of combinations of several of them were studied in vivo and in vitro. In vivo, single-, ΔL4, ΔL5, ΔL6, and double-loop deletions, ΔL4 + ΔL5 and ΔL5 + ΔL6, abolished maltoporin functions, but not the double deletion ΔL4 + ΔL6 and the triple deletion ΔL4 + ΔL5 + ΔL6. While deletion of the central variable portion of loop L9 (ΔL9v) affected maltoporin function only moderately, the combination of ΔL9v with the double deletion of loops L4 and L6 (triple deletion ΔL4 + ΔL6 + ΔL9v) strongly impaired maltoporin function and resulted in sensitivity to large hydrophilic antibiotics without change in channel size as measured in vitro. In vitro, the carbohydrate-binding properties of the different loop mutants were studied in titration experiments using the asymmetric and symmetric addition of the mutant porins and of the carbohydrates to one or both sides of the lipid bilayer membranes. The deletion of loop L9v alone (LamBΔL9v), of two loops L4 and L6 (LamBΔL4 + ΔL6), of three loops L4, L5 and L6 (LamBΔL4 + ΔL5 + ΔL6) or of L4, L6 and L9v (LamBΔL4 + ΔL6 + ΔL9v) had relatively little influence on the carbohydrate-binding properties of the mutant channels, and they had approximately similar binding properties for carbohydrate addition to both sides compared with only one side. The deletion of one of the loops L4 (LamBΔL4) or L6 (LamBΔL6) resulted in an asymmetric carbohydrate binding. The in vivo and in vitro results, together with those of the purification across the starch column,... [ABSTRACT FROM AUTHOR]

After fusion with the N-proximal portion of the outer membrane protein LamB, three β-adrenergic receptors, the human β1- and β2- and turkey β1-adrenergic receptor, were expressed in Escherichia coli with retention of their own specific pharmacological properties. Molecular characterization and localization of the three receptors in bacteria and comparison of the behaviour of each hybrid protein are reported. The bacteria were lysed and fractionated on a sucrose gradient. Saturable [125I]iodocyanopindolol binding activity was found associated mainly with the inner membrane fraction, suggesting that the receptor is correctly folded in this membrane. Binding activity was also found in the outer membrane fraction but varied according to the receptor type. Photoaffinity labeling experiments revealed that the receptors exhibit binding activity only after proteolytic removal of the LamB moiety from the fusion protein. The three hybrid proteins, detected in immunoblots by anti-peptide antibodies, were found mainly in the outer membrane fraction. Each of them exhibited different susceptibility to intrinsic bacterial proteolytic enzymes; sites of proteolytic cleavage were localized by the use of anti-peptide antibodies. The functional expression in E. coli of three β-adrenergic receptors with similar structure but different amino acid sequences suggests that this expression system may be a general feature among similar receptors of the family of G-protein-coupled receptors. The level of expressed binding activity of a given receptor will be within the control of proteolytic degradation processes, depending on the primary sequence of the receptor. Constructions of new hybrid proteins, in combination with expression in protease mutants of E. coli, should help in controlling such processes. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented on the expression and insertion of a monomeric form of protein into the outer membrane of the Escherichia coli genome.

Background: Staphylococcus aureus has emerged as a leading cause of invasive severe diseases with a high rate of morbidity and mortality worldwide. The wide spectrum of clinical manifestations and outcome observed in staphylococcal illness may be a consequence of both microbial factors and variability of the host immune response.Case Presentation: A 14-years old child developed limb ischemia with gangrene following S. aureus bloodstream infection. Histopathology revealed medium-sized arterial vasculitis. The causing strain belonged to the emerging clone CC1-MSSA and numerous pathogenesis-related genes were identified. Patient's genotyping revealed functional variants associated with severe infections. A combination of virulence and host factors might explain this unique severe form of staphylococcal disease.Conclusion: A combination of virulence and genetic host factors might explain this unique severe form of staphylococcal disease. [ABSTRACT FROM AUTHOR]

Recent studies have uncovered the striking commonality of multitasking molecular actors linking metabolism and virulence regulation. Beyond the well known importance of carbohydrate and amino acid metabolism regulators in coordinating metabolic pathways and pathogenesis, we highlight recent major advances linking lipid and nucleic acid pathways to virulence regulation in Staphylococcus aureus. [ABSTRACT FROM AUTHOR]

Summary A putative PEST sequence was recently identified close to the N-terminus of listeriolysin O (LLO), a major virulence factor secreted by the pathogenic Listeria monocytogenes . The deletion of this motif did not affect the secretion and haemolytic activity of LLO, but abolished bacterial virulence. Here, we first tested whether the replacement of the PEST motif of LLO by two different sequences, with either a very high or no PEST score, would affect phagosomal escape, protein stability and, ultimately, the virulence of L. monocytogenes . Then, we constructed LLO mutants with an intact PEST sequence but carrying mutations on either side, or on both sides, of the PEST motif. The properties of these mutants prompted us to construct three LLO mutants carrying single amino acid substitutions in the distal portion of the PEST region (P49A, K50A and P52A; preprotein numbering). Our data demonstrate that the susceptibility of LLO to intracellular proteolytic degradation is not related to the presence of a high PEST score sequence and that the insertion of two residues immediately downstream of the intact PEST sequence is sufficient to impair phagosomal escape and abolish bacterial virulence . Furthermore, we show that single amino acid substitutions in the distal portion of the PEST motif are sufficient to attenuate bacterial ­virulence significantly, unravelling the critical role of this region of LLO in the pathogenesis of L. ­monocytogenes . [ABSTRACT FROM AUTHOR]

A letter to the editor is presented on the production, cellular localization, trimers formation and functional characteristics of Orf538 and Orf414 DNA fragments.

Background: Staphylococcus aureus dominates the lung microbiota of children with cystic fibrosis (CF) and persistent clones are able to establish chronic infection for years, having a direct deleterious impact on lung function. However, in this context, the exact contribution of S. aureus to the decline in respiratory function in children with CF is not elucidated.Methods: To investigate the contribution of persistent S. aureus clones in CF disease, we undertook the analysis of sequential isogenic isolates recovered from 15 young CF patients.Results: Using an air-liquid infection model, we observed a strong correlation between S. aureus adaption in the lung (late isolates), low toxicity, and proinflammatory cytokine secretion. Conversely, early isolates appeared to be highly cytotoxic but did not promote cytokine secretion. We found that cytokine secretion was dependent on staphylococcal protein A (Spa), which was selectively expressed in late compared to early isolates as a consequence of dysfunctional agr quorum-sensing system. Finally, we demonstrated the involvement of TNF-α receptor 1 signaling in the inflammatory response of airway epithelial cells to these lung-adapted S. aureus isolates.Conclusions: Our results suggest an unexpected direct role of bacterial lung adaptation in the progression of chronic lung disease by promoting a proinflammatory response through acquired agr dysfunction. [ABSTRACT FROM AUTHOR]

The bacterial pathogen Listeria monocytogenes invades host cells, ruptures the internalization vacuole, and reaches the cytosol for replication. A high-content small interfering RNA (siRNA) microscopy screen allowed us to identify epithelial cell factors involved in L. monocytogenes vacuolar rupture, including the serine/threonine kinase Taok2. Kinase activity inhibition using a specific drug validated a role for Taok2 in favoring L. monocytogenes cytoplasmic access. Furthermore, we showed that Taok2 recruitment to L. monocytogenes vacuoles requires the presence of pore-forming toxin listeriolysin O. Overall, our study identified the first set of host factors modulating L. monocytogenes vacuolar rupture and cytoplasmic access in epithelial cells. [ABSTRACT FROM AUTHOR]

Metabolic pathways are now considered as intrinsic virulence attributes of pathogenic bacteria and thus represent potential targets for antibacterial strategies. Here we focused on the role of the pentose phosphate pathway (PPP) and its connections with other metabolic pathways in the pathophysiology of Francisella novicida. The involvement of the PPP in the intracellular life cycle of Francisella was first demonstrated by studying PPP inactivating mutants. Indeed, we observed that inactivation of the tktA, rpiA or rpe genes severely impaired intramacrophage multiplication during the first 24 hours. However, time-lapse video microscopy demonstrated that rpiA and rpe mutants were able to resume late intracellular multiplication. To better understand the links between PPP and other metabolic networks in the bacterium, we also performed an extensive proteo-metabolomic analysis of these mutants. We show that the PPP constitutes a major bacterial metabolic hub with multiple connections to glycolysis, the tricarboxylic acid cycle and other pathways, such as fatty acid degradation and sulfur metabolism. Altogether our study highlights how PPP plays a key role in the pathogenesis and growth of Francisella in its intracellular niche. Author summary: Metabolic pathways are intimately associated with the virulence of pathogenic bacteria and are therefore interesting antibacterial targets. Here we focused on the role of the pentose phosphate pathway (PPP) in the intracellular life cycle of the bacterium Francisella novicida. We were able to show, by combining genetic and imaging approaches, that this pathway was a major contributor to the intracellular survival of the bacterium. Complementary proteomics and metabolomics approaches revealed that PPP was at the crossroads of many other metabolic pathways. This work has thus allowed us to better understand the key role played by the PPP in the survival of Francisella in its intracellular niche. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a leading cause of both acute and chronic infections in humans. The importance of the pentose phosphate pathway (PPP) during S. aureus infection is currently largely unexplored. In the current study, we focused on one key PPP enzyme, transketolase (TKT). We showed that inactivation of the unique gene encoding TKT activity in S. aureus USA300 (∆tkt) led to drastic metabolomic changes. Using time-lapse video imaging and mice infection, we observed a major defect of the ∆tkt strain compared with wild-type strain in early intracellular proliferation and in the ability to colonize kidneys. Transcriptional activity of the 2 master regulators sigma B and RpiRc was drastically reduced in the ∆tkt mutant during host cells invasion. The concomitant increased RNAIII transcription suggests that TKT-or a functional PPP-strongly influences the ability of S. aureus to proliferate within host cells by modulating key transcriptional regulators. [ABSTRACT FROM AUTHOR]

Background Chronic lung infection in cystic fibrosis (CF) patients by Staphylococcus aureus is a well-established epidemiological fact. Indeed, S. aureus is the most commonly identified pathogen in the lungs of CF patients. Improving our understanding of the mechanisms associated with the persistence of S. aureus is therefore an important issue. Methods We selected pairs of sequential S. aureus isolates from 3 patients with CF and from 1 patient with non-CF chronic lung disease. We used a combination of genomic, proteomic, and metabolomic approaches with functional assays for in-depth characterization of S. aureus long-term persistence. Results In this study, we show that late S. aureus isolates from CF patients have an increased ability for intracellular survival in CF bronchial epithelial-F508del cells compared to ancestral early isolates. Importantly, the increased ability to persist intracellularly was confirmed for S. aureus isolates within the own-patient F508del epithelial cells. An increased ability to form biofilm was also demonstrated. Furthermore, we identified the underlying genetic modifications that induce altered protein expression profiles and notable metabolic changes. These modifications affect several metabolic pathways and virulence regulators that could constitute therapeutic targets. Conclusions Our results strongly suggest that the intracellular environment might constitute an important niche of persistence and relapse necessitating adapted antibiotic treatments. [ABSTRACT FROM AUTHOR]

Today, in the presence of global warming, understanding how plants respond to drought stress is essential to meet the challenge of developing new cultivars and new irrigation strategies, consistent with the maintenance of crop productivity. In this context, the study of the relation between plants and water is of central interest for modeling their responses to biotic and abiotic constraints. Paradoxically, there are very few direct and noninvasive methods to quantify and measure the level and the flow of water in plants. The present work aims to develop a noninvasive methodology for living plant based on nuclear magnetic resonance (NMR) at low magnetic field and imaging (MRI) to tackle the issue of water quantity in plants. For this purpose, a portable NMR device measuring the signal level at 8 mT was built. This instrument addresses specific challenges such as miniaturization, accessibility, and overheating in order to maintain the plant intact of time over long period. Time dependence of the water content in sorghum plants is reported under abiotic stress as well as the fraction of transpirable soil water and the photosynthesis activity through the leaves. At high magnetic field (9.4 T), T2 maps were acquired on the same sorghum plants at two time points. The combination of these approaches allows us to identify ecophysiological biomarkers of drought stress. One particular interesting result concerns the spatial distribution of water in two anatomically contrasted sorghum genotypes. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is the predominant pathogen in children with cystic fibrosis (CF) in France and, around 80% of them harbored S. aureus in their lungs. This study investigated virulence and antimicrobial resistance-associated genes and within-host evolution polymorphisms in 14 S. aureus persistent clones from 14 chronically infected CF children. For each of the 14 patients, we compared genomes of two isogenic sequential isolates separated by 2–9 years. All isolates were methicillin-sensitive and harbored the immune evasion gene cluster, whereas half of them harbored the enterotoxin gene cluster. Most clones were capsule type 8 (8/14) and accessory gene regulator (agr)-specificity group 1 (9/14). We identified convergent mutations in genes involved in carbohydrate metabolism, cell wall metabolism, genetic information processing and adhesion, which are likely to play important role in intracellular invasion and persistence. Further explorations relying notably on proteomics will contribute to improve our understanding of the mechanisms at play in the striking long-term persistence ability of S. aureus. [ABSTRACT FROM AUTHOR]

Intracellular multiplication and dissemination of the infectious bacterial pathogen F rancisella tularensis implies the utilization of multiple host-derived nutrients. Here, we demonstrate that gluconeogenesis constitutes an essential metabolic pathway in F rancisella pathogenesis. Indeed, inactivation of gene glp X, encoding the unique fructose 1,6-bisphosphatase of F rancisella, severely impaired bacterial intracellular multiplication when cells were supplemented by gluconeogenic substrates such as glycerol or pyruvate. The Δ glp X mutant also showed a severe virulence defect in the mouse model, confirming the importance of this pathway during the in vivo life cycle of the pathogen. Isotopic profiling revealed the major role of the Embden- Meyerhof (glycolysis) pathway in glucose catabolism in F rancisella and confirmed the importance of glp X in gluconeogenesis. Altogether, the data presented suggest that gluconeogenesis allows F rancisella to cope with the limiting glucose availability it encounters during its infectious cycle by relying on host amino acids. Hence, targeting the gluconeogenic pathway might constitute an interesting therapeutic approach against this pathogen. [ABSTRACT FROM AUTHOR]

As commonly seen in monoderm bacteria, Listeria monocytogenes possesses multiple membrane-bound signal peptidases of Type I (SPases I) called SipX, SipY and SipZ. In order to decipher their respective contribution in an integrated and global view, the complement of the secretome corresponding to the exoproteome was resolved by two-dimensional gel electrophoresis (2-DE). This was performed for L. monocytogenes sipX − , sipY − , sipZ − single mutants, as well as for Δ sipXY and Δ sipYZ double mutants, and then compared to that of the wild type strain. Remarkably, the amounts of listeriolysin O (LLO), phosphatidylcholine phospholipase C (PlcB) and zinc metalloproteinase Mpl in the extracellular milieu were significantly decreased upon inactivation of SipZ. For the majority of the Sec-secreted exoproteins identified, protein secretion was not affected by the inactivation of one or two of the SPases I, supporting the concept that the three SPases I have overlapping specificities for the cleavage of the signal peptides. The current study reveals that the role of SipZ as the major SPase I of L. monocytogenes applies only to a small subset of the secreted exoproteins. Rather than absolute, the notion of major and minor SPases thus appears to be relative. In addition to new insight into bacterial physiology, this investigation of the contribution of the SPases I to the exoproteome of L. monocytogenes paves the way for further characterization of other complements of the secretome under various environmental conditions. Biological significance L. monocytogenes encodes three orthologous signal peptidases of Type I (SPases I). SipZ improves the secretion efficiency for a subset of extracellular virulence factors. Multiple SPases I are functionally redundant for the majority of the Sec-secreted exoproteins of L. monocytogenes . The concepts of major and minor SPases are not absolute but relative. [ABSTRACT FROM AUTHOR]