Streptococcal pyrogenic exotoxin B cleaves GSDMA and triggers pyroptosis.

Gasdermins, a family of five pore-forming proteins (GSDMA-GSDME) in humans expressed predominantly in the skin, mucosa and immune sentinel cells, are key executioners of inflammatory cell death (pyroptosis), which recruits immune cells to infection sites and promotes protective immunity1,2. Pore formation is triggered by gasdermin cleavage1,2. Although the proteases that activate GSDMB, C, D and E have been identified, how GSDMA-the dominant gasdermin in the skin-is activated, remains unknown. Streptococcus pyogenes, also known as group A Streptococcus (GAS), is a major skin pathogen that causes substantial morbidity and mortality worldwide3. Here we show that the GAS cysteine protease SpeB virulence factor triggers keratinocyte pyroptosis by cleaving GSDMA after Gln246, unleashing an active N-terminal fragment that triggers pyroptosis. Gsdma1 genetic deficiency blunts mouse immune responses to GAS, resulting in uncontrolled bacterial dissemination and death. GSDMA acts as both a sensor and substrate of GAS SpeB and as an effector to trigger pyroptosis, adding a simple one-molecule mechanism for host recognition and control of virulence of a dangerous microbial pathogen.

Virulence attributes of successful methicillin-resistant Staphylococcus aureus lineages.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of severe and often fatal infections. MRSA epidemics have occurred in waves, whereby a previously successful lineage has been replaced by a more fit and better adapted lineage. Selection pressures in both hospital and community settings are not uniform across the globe, which has resulted in geographically distinct epidemiology. This review focuses on the mechanisms that trigger the establishment and maintenance of current, dominant MRSA lineages across the globe. While the important role of antibiotic resistance will be mentioned throughout, factors which influence the capacity of S. aureus to colonize and cause disease within a host will be the primary focus of this review. We show that while MRSA possesses a diverse arsenal of toxins including alpha-toxin, the success of a lineage involves more than just producing toxins that damage the host. Success is often attributed to the acquisition or loss of genetic elements involved in colonization and niche adaptation such as the arginine catabolic mobile element, as well as the activity of regulatory systems, and shift metabolism accordingly (e.g., the accessory genome regulator, agr). Understanding exactly how specific MRSA clones cause prolonged epidemics may reveal targets for therapies, whereby both core (e.g., the alpha toxin) and acquired virulence factors (e.g., the Panton-Valentine leukocidin) may be nullified using anti-virulence strategies.

Increased intracellular persulfide levels attenuate HlyU-mediated hemolysin transcriptional activation in Vibrio cholerae.

The vertebrate host's immune system and resident commensal bacteria deploy a range of highly reactive small molecules that provide a barrier against infections by microbial pathogens. Gut pathogens, such as Vibrio cholerae, sense and respond to these stressors by modulating the expression of exotoxins that are crucial for colonization. Here, we employ mass spectrometry-based profiling, metabolomics, expression assays, and biophysical approaches to show that transcriptional activation of the hemolysin gene hlyA in V. cholerae is regulated by intracellular forms of sulfur with sulfur-sulfur bonds, termed reactive sulfur species (RSS). We first present a comprehensive sequence similarity network analysis of the arsenic repressor superfamily of transcriptional regulators, where RSS and hydrogen peroxide sensors segregate into distinct clusters of sequences. We show that HlyU, transcriptional activator of hlyA in V. cholerae, belongs to the RSS-sensing cluster and readily reacts with organic persulfides, showing no reactivity or DNA dissociation following treatment with glutathione disulfide or hydrogen peroxide. Surprisingly, in V. cholerae cell cultures, both sulfide and peroxide treatment downregulate HlyU-dependent transcriptional activation of hlyA. However, RSS metabolite profiling shows that both sulfide and peroxide treatment raise the endogenous inorganic sulfide and disulfide levels to a similar extent, accounting for this crosstalk, and confirming that V. cholerae attenuates HlyU-mediated activation of hlyA in a specific response to intracellular RSS. These findings provide new evidence that gut pathogens may harness RSS-sensing as an evolutionary adaptation that allows them to overcome the gut inflammatory response by modulating the expression of exotoxins.

Caspase-1-driven neutrophil pyroptosis and its role in host susceptibility to Pseudomonas aeruginosa.

Multiple regulated neutrophil cell death programs contribute to host defense against infections. However, despite expressing all necessary inflammasome components, neutrophils are thought to be generally defective in Caspase-1-dependent pyroptosis. By screening different bacterial species, we found that several Pseudomonas aeruginosa (P. aeruginosa) strains trigger Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P. aeruginosa enhanced neutrophil death to Caspase-1-dependent pyroptosis, suggesting that these exotoxins interfere with this pathway. Mechanistically, P. aeruginosa Flagellin activates the NLRC4 inflammasome, which supports Caspase-1-driven interleukin (IL)-1ß secretion and Gasdermin D (GSDMD)-dependent neutrophil pyroptosis. Furthermore, P. aeruginosa-induced GSDMD activation triggers Calcium-dependent and Peptidyl Arginine Deaminase-4-driven histone citrullination and translocation of neutrophil DNA into the cell cytosol without inducing extracellular Neutrophil Extracellular Traps. Finally, we show that neutrophil Caspase-1 contributes to IL-1ß production and susceptibility to pyroptosis-inducing P. aeruginosa strains in vivo. Overall, we demonstrate that neutrophils are not universally resistant for Caspase-1-dependent pyroptosis.

Exotoxin S secreted by internalized Pseudomonas aeruginosa delays lytic host cell death.

The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.

Exploring the pathogenicity of Mycoplasma pneumoniae: Focus on community-acquired respiratory distress syndrome toxins.

Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX) is a unique exotoxin produced by Mycoplasma pneumoniae (MP) and has been confirmed to possess ADP-ribosyltransferase (ART) and vacuolating activities. CARDS TX binds to receptors on the surfaces of mammalian cells followed by entry into the cells through clathrin-mediated endocytosis, and exerts cytotoxic effects by undergoing retrograde transport and finally cleavage on endosomes and cellular organelles. In addition, CARDS TX can trigger severe inflammatory reactions resulting in airway dysfunction, producing allergic inflammation and asthma-like conditions. As a newly discovered virulence factor of MP, CARDS TX has been extensively studied in recent years. As resistance to macrolide drugs has increased significantly in recent years and there is no vaccine against MP, the development of a vaccine targeting CARDS TX is considered a potential preventive measure. This review focuses on recent studies and insights into this toxin, providing directions for a better understanding of MP pathogenesis and treatment. IMPORTANCE: A serious hazard to worldwide public health in recent years, Mycoplasma pneumoniae (MP) is a prominent bacterium that causes community-acquired pneumonia (CAP) in hospitalized children. Due to their high prevalence and fatality rates, MP infections often cause both respiratory illnesses and extensive extrapulmonary symptoms. It has recently been shown that MP produces a distinct exotoxin known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). Mycoplasma pneumoniae pneumonia (MPP)-like tissue injury is caused by this toxin because it has both ADP-ribosyltransferase and vacuolating properties. A better knowledge of MP etiology and therapy is provided by this review, which focuses on latest research and insights into this toxin.

Exploration of compounds to inhibit the Panton-Valentine leukocidin of Staphylococcus aureus.

The Panton-Valentine leukocidin (PVL) of Staphylococcus aureus is associated with necrotizing infections. After binding to complement 5a receptor (C5aR/CD88) and CD45 it causes cytolysis in polymorphonuclear neutrophils (PMNs) as well as inflammasome activation in monocytes. The objective of this study was to test if (ant)agonists of C5aR and CD45 can attenuate the effect of PVL on PMNs and monocytes. We tested the effect of various concentrations of six C5aR (ant)agonists (avacopan, BM213, DF2593A, JPE-1375, PMX205 and W-54011) and one CD45 antagonist (NQ301) to attenuate the cytotoxic effect of PVL on human PMNs and monocytes in vitro. Shifts in the half-maximal effective concentration (EC50) of PVL to achieve a cytotoxic effect on PMNs and modulation of inflammatory cytokine response from monocytes were determined by flow cytometry and IL-1ß detection. Pre-treatment of PMNs with avacopan, PMX205 and W-54,011 resulted in 3.6- to 4.3-fold shifts in the EC50 for PVL and were able to suppress IL-1ß secretion by human monocytes in the presence of PVL. BM213, DF2593A and NQ301 were unable to change the susceptibility of PMNs towards PVL or reduce inflammasome activation in monocytes. Avacopan, PMX205 and W-54,011 showed protection against PVL-induced cytotoxicity and suppressed IL-1ß secretion by monocytes. Clinical studies are needed to prove whether these substances can be used therapeutically as repurposed drugs.

Molecular insights into mechanisms of GPCR hijacking by Staphylococcus aureus.

Atypical chemokine receptor 1 (ACKR1) is a G protein-coupled receptor (GPCR) targeted by Staphylococcus aureus bicomponent pore-forming leukotoxins to promote bacterial growth and immune evasion. Here, we have developed an integrative molecular pharmacology and structural biology approach in order to characterize the effect of leukotoxins HlgA and HlgB on ACKR1 structure and function. Interestingly, using cell-based assays and native mass spectrometry, we found that both components HlgA and HlgB compete with endogenous chemokines through a direct binding with the extracellular domain of ACKR1. Unexpectedly, hydrogen/deuterium exchange mass spectrometry analysis revealed that toxin binding allosterically modulates the intracellular G protein-binding domain of the receptor, resulting in dissociation and/or changes in the architecture of ACKR1-Gαi1 protein complexes observed in living cells. Altogether, our study brings important molecular insights into the initial steps of leukotoxins targeting a host GPCR.

Membrane Interaction Characteristics of the RTX Toxins and the Cholesterol-Dependence of Their Cytolytic/Cytotoxic Activity.

RTX toxins are important virulence factors produced by a wide range of Gram-negative bacteria. They are secreted as water-soluble proteins that are able to bind to the host cell membrane and insert hydrophobic segments into the lipid bilayer that ultimately contribute to the formation of transmembrane pores. Ion diffusion through these pores leads then to cytotoxic and cytolytic effects on the hosts. Several reports have evidenced that the binding of several RTX toxins to the target cell membrane may take place through a high-affinity interaction with integrins of the ß2 family that is highly expressed in immune cells of the myeloid lineage. However, at higher toxin doses, cytotoxicity by most RTX toxins has been observed also on ß2-deficient cells in which toxin binding to the cell membrane has been proposed to occur through interaction with glycans of glycosylated lipids or proteins present in the membrane. More recently, cumulative pieces of evidence show that membrane cholesterol is essential for the mechanism of action of several RTX toxins. Here, we summarize the most important aspects of the RTX toxin interaction with the target cell membrane, including the cholesterol dependence, the recent identification in the sequences of several RTX toxins of linear motifs coined as the Cholesterol Recognition/interaction Amino acid Consensus (CRAC), and the reverse or mirror CARC motif, which is involved in the toxin-cholesterol interaction.

SarS Is a Repressor of Staphylococcus aureus Bicomponent Pore-Forming Leukocidins.

Staphylococcus aureus is a successful pathogen that produces a wide range of virulence factors that it uses to subvert and suppress the immune system. These include the bicomponent pore-forming leukocidins. How the expression of these toxins is regulated is not completely understood. Here, we describe a screen to identify transcription factors involved in the regulation of leukocidins. The most prominent discovery from this screen is that SarS, a known transcription factor which had previously been described as a repressor of alpha-toxin expression, was found to be a potent repressor of leukocidins LukED and LukSF-PV. We found that inactivating sarS resulted in increased virulence both in an ex vivo model using primary human neutrophils and in an in vivo infection model in mice. Further experimentation revealed that SarS represses leukocidins by serving as an activator of Rot, a critical repressor of toxins, as well as by directly binding and repressing the leukocidin promoters. By studying contemporary clinical isolates, we identified naturally occurring mutations in the sarS promoter that resulted in overexpression of sarS and increased repression of leukocidins in USA300 bloodstream clinical isolates. Overall, these data establish SarS as an important repressor of leukocidins and expand our understanding of how these virulence factors are being regulated in vitro and in vivo by S. aureus.

Revealing the Achilles heel of bacterial toxins.

In addition to having antimicrobial properties, defensins inactivate various structurally unrelated bacterial toxins by a yet unknown manner. In this issue of Immunity, Kudryashova et al. (2014b) provide insights into mechanisms by which human ?-defensins destabilize and inactivate bacterial toxins.

Human defensins facilitate local unfolding of thermodynamically unstable regions of bacterial protein toxins.

Defensins are short cationic, amphiphilic, cysteine-rich peptides that constitute the front-line immune defense against various pathogens. In addition to exerting direct antibacterial activities, defensins inactivate several classes of unrelated bacterial exotoxins. To date, no coherent mechanism has been proposed to explain defensins' enigmatic efficiency toward various toxins. In this study, we showed that binding of neutrophil ?-defensin HNP1 to affected bacterial toxins caused their local unfolding, potentiated their thermal melting and precipitation, exposed new regions for proteolysis, and increased susceptibility to collisional quenchers without causing similar effects on tested mammalian structural and enzymatic proteins. Enteric ?-defensin HD5 and ?-defensin hBD2 shared similar toxin-unfolding effects with HNP1, albeit to different degrees. We propose that protein susceptibility to inactivation by defensins is contingent to their thermolability and conformational plasticity and that defensin-induced unfolding is a key element in the general mechanism of toxin inactivation by human defensins.

Transcriptional Regulators Controlling Virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a pathogen capable of colonizing virtually every human tissue. The host colonization competence and versatility of this pathogen are powered by a wide array of virulence factors necessary in different steps of the infection process. This includes factors involved in bacterial motility and attachment, biofilm formation, the production and secretion of extracellular invasive enzymes and exotoxins, the production of toxic secondary metabolites, and the acquisition of iron. Expression of these virulence factors during infection is tightly regulated, which allows their production only when they are needed. This process optimizes host colonization and virulence. In this work, we review the intricate network of transcriptional regulators that control the expression of virulence factors in P. aeruginosa, including one- and two-component systems and σ factors. Because inhibition of virulence holds promise as a target for new antimicrobials, blocking the regulators that trigger the production of virulence determinants in P. aeruginosa is a promising strategy to fight this clinically relevant pathogen.

Development of anti DLL4 Nanobody fused to truncated form of Pseudomonas exotoxin: As a novel immunotoxin to inhibit of cell proliferation and neovascularization.

Targeted tumor therapy is an attractive approach for cancer treatment. Delta-like ligand 4 (DLL4) is overexpressed in tumor vasculature and plays a pivotal role in tumor neovascular development and angiogenesis during tumor progression. Immunotoxins due to their superior cell-killing ability and the relative simplicity of their preparation, have great potential in the clinical treatment of cancer. The aim of this study was to develop a novel immunotoxin against DLL4 as a cell cytotoxic agent and angiogenesis maturation inhibitor. In present study, an immunotoxin, named DLL4Nb-PE, in which a Nanobody as targeting moiety fused to the Pseudomonas exotoxin A (PE) was constructed, expressed and assessed by SDS-PAGE, western blotting, ELISA and flowcytometry. The functional assessment was carried out via MTT, apoptosis and chicken chorioallantoic membrane (CAM) assays. It was demonstrated DLL4Nb-PE specifically binds to DLL4 and recognizes DLL4-expressing MKN cells. The cytotoxicity assays showed that this molecule could induce apoptosis and kill DLL4 positive MKN cells. In addition, it inhibited neovascularization in the chicken chorioallantoic membrane. Our findings indicate designed anti-DLL4 immunotoxin has valuable potential for application to the treatment of tumors with high DLL4 expression.

Staphylococcus aureus Panton-Valentine Leukocidin triggers an alternative NETosis process targeting mitochondria.

Panton-Valentine Leukocidin (PVL) is a bicomponent leukotoxin produced by 3%-10% of clinical Staphylococcus aureus (SA) strains involved in the severity of hospital and community-acquired infections. Although PVL was long known as a pore-forming toxin, recent studies have challenged the formation of a pore at the plasma membrane, while its endocytosis and the exact mode of action remain to be defined. In vitro immunolabeling of human neutrophils shows that Neutrophil Extracellular Traps (NETosis) is triggered by the action of purified PVL, but not by Gamma hemolysin CB (HlgCB), a structurally similar SA leukotoxin. PVL causes the ejection of chromatin fibers (NETs) decorated with antibacterial peptides independently of the NADPH oxidase oxidative burst. Leukotoxin partially colocalizes with mitochondria and enhances the production of reactive oxygen species from these organelles, while showing an increased autophagy, which results unnecessary for NETs ejection. PVL NETosis is elicited through Ca2+ -activated SK channels and Myeloperoxidase activity but is abolished by Allopurinol pretreatment of neutrophils. Moreover, massive citrullination of the histone H3 is performed by peptidyl arginine deiminases. Inhibition of this latter enzymes fails to abolish NET extrusion. Unexpectedly, PVL NETosis does not seem to involve Src kinases, which is the main kinase family activated downstream the binding of PVL F subunit to CD45 receptor, while the specific kinase pathway differs from the NADPH oxidase-dependent NETosis. PVL alone causes a different and specific form of NETosis that may rather represent a bacterial strategy conceived to disarm and disrupt the immune response, eventually allowing SA to spread.

Secretome analysis of an environmental isolate Enterobacter sp. S-33 identifies proteins related to pathogenicity.

Enterobacter species are responsible for causing infections of the lower respiratory tract, urinary tract, meninges, etc. Proteins secreted by these species may act as determinants of host-pathogen interaction and play a role in virulence. Among the secreted proteins, the Type VI secretion system (T6SS) acts as a molecular nanomachine to deliver many effector proteins directly into prey cells in a contact-dependent manner. The secreted proteins may provide an idea for the interaction of bacteria to their environment and an understanding of the role of these proteins for their role in bacterial physiology and behaviour. Therefore, aim of this study was to characterize the secreted proteins in the culture supernatant by a T6SS bacterium Enterobacter sp. S-33 using nano-LC-MS/MS tool. Using a combined mass spectrometry and bioinformatics approach, we identified a total of 736 proteins in the secretome. Bioinformatics analysis predicting subcellular localization identified 110 of the secreted proteins possessed signal sequences. By gene ontology analysis, more than 80 proteins of the secretome were classified into biological or molecular functions. More than 20 percent of secretome proteins were virulence proteins including T6SS proteins, proteins involved in adherence and fimbriae formation, molecular chaperones, outer membrane proteins, serine proteases, antimicrobial, biofilm, exotoxins, etc. In summary, the results of the present study of the S-33 secretome provide a basis for understanding the possible pathogenic mechanisms and future investigation by detailed experimental approach will provide a confirmation of secreted virulence proteins in the exact role of virulence using the in vivo model.

Crp/fnr family protein binds to promoters of atxA and sodmn genes that regulate the expression of exotoxins in Bacillus anthracis.

Bacillus anthracis produces a tripartite exotoxin, which is regulated by AtxA. Sodmn is constitutively expressed during invasion. Crp/Fnr family transcriptional regulators are known to bind promoters of toxin regulators as well as constitutively expressed genes during pathogenesis. B. anthracis fnr gene was cloned, over-expressed in E. coli and recombinant protein was purified. Oligomeric nature of recombinant rFnr protein was studied by diamide treatment and DTT reduction. DNA binding of rFnr protein was studied by EMSA. We observed that rFnr exists in both monomeric and oligomeric forms. It was found that rFnr was able to oligomerize after diamide treatment which was reversible through DTT reduction. Promoter regions of atxA and sodmn show binding to monomeric form of rFnr, however, dimeric form was unable to bind. Fnr might be playing a role in regulation of toxin gene expression via regulation of atxA gene. It can also be involved in regulation of pathogenesis by regulating the sodmn expression. Oligomerization can act as an ON/OFF switch for the Fnr mediated regulation.

A case of acute poststreptococcal glomerulonephritis complicated by interstitial nephritis related to streptococcal pyrogenic exotoxin B.

This paper presents the case of a patient who developed acute kidney injury and nephrotic syndrome following streptococcal cutaneous infection. He presented with microhematuria, severe proteinuria and systemic edema 5 days after infection. Blood examination showed elevated creatinine level, hypocomplementemia, and elevated anti-streptolysin O level. Renal biopsy revealed endocapillary proliferative glomerulonephritis with tubulointerstitial nephritis (TIN). Immunofluorescence revealed C3-dominant glomerular staining, while electron microscopy showed hump-shaped subepithelial deposits. The patient was therefore diagnosed with poststreptococcal glomerulonephritis. The unique histological feature was C3 deposition in the tubular basement membrane (TBM), in which we detected streptococcal pyrogenic exotoxin B (SpeB), a nephritogenic antigen produced by streptococci. No nephritis-associated plasmin receptor or plasmin activity was evident in the TBM. These nephritogenic antigens and upregulation of plasmin activity were observed in glomeruli. This case suggests that TIN after poststreptococcal infection might be partially attributable to SpeB toxicity.

Panton-Valentine leucocidin-producing Staphylococcus aureus: a clinical review.

Panton-Valentine leucocidin (PVL) is a virulence factor produced by certain strains of Staphylococcus aureus (SA). Through its cytolytic action on the cell membranes of human polymorphonuclear neutrophils, PVL causes a range of pathologies collectively known as PVL-SA disease. The hallmark clinical signs of PVL-SA are recurrent boils and necrotizing skin and soft tissue infections (SSTIs) in otherwise healthy patients; however, it can lead to more severe and invasive presentations, including necrotizing haemorrhagic pneumonia, necrotizing fasciitis and purpura fulminans. Young adults with minimal previous exposure to healthcare settings tend to be at highest risk for acquiring PVL-SA disease, with close physical contact playing a central role in disease transmission. The prevalence of PVL-SA varies globally; however, this is often underestimated owing to a lack of routine PVL testing. In the UK, PVL-positive SA isolates have been rising over the past decade alongside an increasing prevalence of multidrug resistance in larger cities. This review article aims to raise awareness of the PVL toxin, to aid clinicians with diagnostic pointers and to provide guidance with treatment, with an emphasis on the need for further population-based studies.

Aggregatibacter actinomycetemcomitans colonization and persistence in a primate model.

Aggregatibacter actinomycetemcomitans is associated with aggressive periodontitis resulting in premature tooth loss in adolescents. Tooth adherence and biofilm persistence are prerequisites for survival in the oral domain. Here, using a rhesus monkey model, 16S rRNA sequencing, and weighted network analysis, we assessed colonization of A. actinomycetemcomitans variants and ascertained microbial interactions in biofilm communities. Variants in A. actinomycetemcomitans leukotoxin (ltx) were created, labeled, inoculated, and compared with their progenitor strain for in vivo colonization. Samples of tooth-related plaque were assessed for colonization at baseline and after debridement and inoculation of labeled strains. Null, minimal, and hyper-Ltx-producing strains were created and assessed for hydroxyapatite binding and biofilm formation in vitro. Ltx-hyperproducing strains colonized with greater prevalence and at higher levels than wild type or ltx mutants (P = 0.05). Indigenous and inoculated A. actinomycetemcomitans strains that attached were associated with lactate-producing species (i.e., Leptotrichia, Abiotrophia, and Streptoccocci). A. actinomycetemcomitans was found at 0.13% of the total flora at baseline and at 0.05% 4 wk after inoculation. In vivo data were supported by in vitro results. We conclude that hyper-Ltx production affords these strains with an attachment advantage providing a foothold for competition with members of the indigenous microbiota. Increased attachment can be linked to ltx gene expression and up-regulation of adherence-associated genes. Growth of attached A. actinomycetemcomitans in vivo was enhanced by lactate availability due to consorting species. These associations provide A. actinomycetemcomitans with the constituents required for its colonization and survival in the complex and competitive oral environment.