Lactobacilli Downregulate Transcription Factors in Helicobacter pylori That Affect Motility, Acid Tolerance and Antimicrobial Peptide Survival.

Helicobacter pylori infection triggers inflammation that may lead to gastritis, stomach ulcers and cancer. Probiotic bacteria, such as Lactobacillus, have been of interest as treatment options, however, little is known about the molecular mechanisms of Lactobacillus-mediated inhibition of H. pylori pathogenesis. In this work, we investigated the effect of Lactobacillus culture supernatants, so-called conditioned medium (CM), from two gastric isolates, L. gasseri and L. oris, on the expression of transcriptional regulators in H. pylori. Among the four known two-component systems (TCSs), i.e., ArsRS, FlgRS, CheAY and CrdRS, the flagellar regulator gene flgR and the acid resistance associated arsS gene were down-regulated by L. gasseri CM, whereas expression of the other TCS-genes remained unaffected. L. gasseri CM also reduced the motility of H. pylori, which is in line with reduced flgR expression. Furthermore, among six transcription factors of H. pylori only the ferric uptake regulator gene fur was regulated by L. gasseri CM. Deletion of fur further led to dramatically increased sensitivity to the antimicrobial peptide LL-37. Taken together, the results highlight that released/secreted factors of some lactobacilli, but not all, downregulate transcriptional regulators involved in motility, acid tolerance and LL-37 sensitivity of H. pylori.

Lactate-Induced Dispersal of Neisseria meningitidis Microcolonies Is Mediated by Changes in Cell Density and Pilus Retraction and Is Influenced by Temperature Change.

Neisseria meningitidis is the etiologic agent of meningococcal meningitis and sepsis. Initial colonization of meningococci in the upper respiratory tract epithelium is crucial for disease development. The colonization occurs in several steps and expression of type IV pili (Tfp) is essential for both attachment and microcolony formation of encapsulated bacteria. Previously, we have shown that host-derived lactate induces synchronized dispersal of meningococcal microcolonies. In this study, we demonstrated that lactate-induced dispersal is dependent on bacterial concentration but not on the quorum-sensing system autoinducer-2 or the two-component systems NarP/NarQ, PilR/PilS, NtrY/NtrX, and MisR/MisS. Further, there were no changes in expression of genes related to assembly, elongation, retraction, and modification of Tfp throughout the time course of lactate induction. By using pilT and pptB mutants, however, we found that lactate-induced dispersal was dependent on PilT retraction but not on phosphoglycerol modification of Tfp even though the PptB activity was important for preventing reaggregation postdispersal. Furthermore, protein synthesis was required for lactate-induced dispersal. Finally, we found that at a lower temperature, lactate-induced dispersal was delayed and unsynchronized, and bacteria reformed microcolonies. We conclude that lactate-induced microcolony dispersal is dependent on bacterial concentration, PilT-dependent Tfp retraction, and protein synthesis and is influenced by environmental temperature.

Modulation of Human Beta-Defensin 2 Expression by Pathogenic Neisseria meningitidis and Commensal Lactobacilli.

Antimicrobial peptides (AMPs) play an important role in the defense against pathogens by targeting and killing invading microbes. Some pathogenic bacteria have been shown to negatively regulate AMP expression, while several commensals may induce AMP expression. The expression of certain AMPs, such as human beta-defensin 2 (hBD2), can be induced via nuclear factor NF-κB, which, in turn, is negatively controlled by tumor necrosis factor alpha-induced protein 3 (TNFAIP3, or A20). In this work, we examined the expression of hBD1 and hBD2 during coincubation of pharyngeal epithelial cells with pathogenic Neisseria meningitidis and commensal lactobacilli. The Lactobacillus strains induced hBD2 expression in human pharyngeal cells, while the pathogen N. meningitidis did not. In coincubation experiments, meningococci were able to dampen the AMP expression induced by lactobacilli. We found that N. meningitidis induced the NF-κB inhibitor A20. Further, RNA silencing of A20 resulted in increased hBD2 expression after meningococcal infection. Since it is known that induction of A20 reduces NF-κB activity and thus hBD2 levels, meningococcal-mediated A20 induction could be a way for the pathogen to dampen AMP expression. Finally, treatment of N. meningitidis and lactobacilli with synthetic hBD2 reduced N. meningitidis viability more efficiently than Lactobacillus reuteri, explaining why maintaining low AMP levels is important for the survival of the pathogen.

Host cell-derived lactate functions as an effector molecule in Neisseria meningitidis microcolony dispersal.

The development of meningococcal disease, caused by the human pathogen Neisseria meningitidis, is preceded by the colonization of the epithelial layer in the nasopharynx. After initial adhesion to host cells meningococci form aggregates, through pilus-pilus interactions, termed microcolonies from which the bacteria later detach. Dispersal from microcolonies enables access to new colonization sites and facilitates the crossing of the cell barrier; however, this process is poorly understood. In this study, we used live-cell imaging to investigate the process of N. meningitidis microcolony dispersal. We show that direct contact with host cells is not required for microcolony dispersal, instead accumulation of a host-derived effector molecule induces microcolony dispersal. By using a host-cell free approach, we demonstrated that lactate, secreted from host cells, initiate rapid dispersal of microcolonies. Interestingly, metabolic utilization of lactate by the bacteria was not required for induction of dispersal, suggesting that lactate plays a role as a signaling molecule. Furthermore, Neisseria gonorrhoeae microcolony dispersal could also be induced by lactate. These findings reveal a role of host-secreted lactate in microcolony dispersal and virulence of pathogenic Neisseria.

Neisseria meningitidis Polynucleotide Phosphorylase Affects Aggregation, Adhesion, and Virulence.

Neisseria meningitidis autoaggregation is an important step during attachment to human cells. Aggregation is mediated by type IV pili and can be modulated by accessory pilus proteins, such as PilX, and posttranslational modifications of the major pilus subunit PilE. The mechanisms underlying the regulation of aggregation remain poorly characterized. Polynucleotide phosphorylase (PNPase) is a 3'-5' exonuclease that is involved in RNA turnover and the regulation of small RNAs. In this study, we biochemically confirm that NMC0710 is the N. meningitidis PNPase, and we characterize its role in N. meningitidis pathogenesis. We show that deletion of the gene encoding PNPase leads to hyperaggregation and increased adhesion to epithelial cells. The aggregation induced was found to be dependent on pili and to be mediated by excessive pilus bundling. PNPase expression was induced following bacterial attachment to human cells. Deletion of PNPase led to global transcriptional changes and the differential regulation of 469 genes. We also demonstrate that PNPase is required for full virulence in an in vivo model of N. meningitidis infection. The present study shows that PNPase negatively affects aggregation, adhesion, and virulence in N. meningitidis.

Lactobacilli Reduce Helicobacter pylori Attachment to Host Gastric Epithelial Cells by Inhibiting Adhesion Gene Expression.

The human gastrointestinal tract, including the harsh environment of the stomach, harbors a large variety of bacteria, of which Lactobacillus species are prominent members. The molecular mechanisms by which species of lactobacilli interfere with pathogen colonization are not fully characterized. In this study, we aimed to study the effect of lactobacillus strains upon the initial attachment of Helicobacter pylori to host cells. Here we report a novel mechanism by which lactobacilli inhibit adherence of the gastric pathogen H. pylori In a screen with Lactobacillus isolates, we found that only a few could reduce adherence of H. pylori to gastric epithelial cells. Decreased attachment was not due to competition for space or to lactobacillus-mediated killing of the pathogen. Instead, we show that lactobacilli act on H. pylori directly by an effector molecule that is released into the medium. This effector molecule acts on H. pylori by inhibiting expression of the adhesin-encoding gene sabA Finally, we verified that inhibitory lactobacilli reduced H. pylori colonization in an in vivo model. In conclusion, certain Lactobacillus strains affect pathogen adherence by inhibiting sabA expression and thereby reducing H. pylori binding capacity.

Nuclear trafficking, histone cleavage and induction of apoptosis by the meningococcal App and MspA autotransporters.

Neisseria meningitidis, a major cause of bacterial meningitis and septicaemia, secretes multiple virulence factors, including the adhesion and penetration protein (App) and meningococcal serine protease A (MspA). Both are conserved, immunogenic, type Va autotransporters harbouring S6-family serine endopeptidase domains. Previous work suggested that both could mediate adherence to human cells, but their precise contribution to meningococcal pathogenesis was unclear. Here, we confirm that App and MspA are in vivo virulence factors since human CD46-expressing transgenic mice infected with meningococcal mutants lacking App, MspA or both had improved survival rates compared with mice infected with wild type. Confocal imaging showed that App and MspA were internalized by human cells and trafficked to the nucleus. Cross-linking and enzyme-linked immuno assay (ELISA) confirmed that mannose receptor (MR), transferrin receptor 1 (TfR1) and histones interact with MspA and App. Dendritic cell (DC) uptake could be blocked using mannan and transferrin, the specific physiological ligands for MR and TfR1, whereas in vitro clipping assays confirmed the ability of both proteins to proteolytically cleave the core histone H3. Finally, we show that App and MspA induce a dose-dependent increase in DC death via caspase-dependent apoptosis. Our data provide novel insights into the roles of App and MspA in meningococcal infection.

Characterization of motility and piliation in pathogenic Neisseria.

BACKGROUND: The type IV pili (Tfp) of pathogenic Neisseria (i.e., N. gonorrhoeae and N. meningitidis) are essential for twitching motility. Tfp retraction, which is dependent on the ATPase PilT, generates the forces that move bacteria over surfaces. Neisseria motility has mainly been studied in N. gonorrhoeae whereas the motility of N. meningitidis has not yet been characterized. RESULTS: In this work, we analyzed bacterial motility and monitored Tfp retraction using live-cell imaging of freely moving bacteria. We observed that N. meningitidis moved over surfaces at an approximate speed of 1.6 µm/s, whereas N. gonorrhoeae moved with a lower speed (1.0 µm/s). An alignment of the meningococcal and gonococcal pilT promoters revealed a conserved single base pair variation in the -10 promoter element that influence PilT expression. By tracking mutants with altered pilT expression or pilE sequence, we concluded that the difference in motility speed was independent of both. Live-cell imaging using total internal reflection fluorescence microscopy demonstrated that N. gonorrhoeae more often moved with fewer visible retracting filaments when compared to N. meningitidis. Correspondingly, meningococci also displayed a higher level of piliation in transmission electron microscopy. Nevertheless, motile gonococci that had the same number of filaments as N. meningitidis still moved with a lower speed. CONCLUSIONS: These data reveal differences in both speed and piliation between the pathogenic Neisseria species during twitching motility, suggesting a difference in Tfp-dynamics.

Uncovering the mechanism of trapping and cell orientation during Neisseria gonorrhoeae twitching motility.

Neisseria gonorrheae bacteria are the causative agent of the second most common sexually transmitted infection in the world. The bacteria move on a surface by means of twitching motility. Their movement is mediated by multiple long and flexible filaments, called type IV pili, that extend from the cell body, attach to the surface, and retract, thus generating a pulling force. Moving cells also use pili to aggregate and form microcolonies. However, the mechanism by which the pili surrounding the cell body work together to propel bacteria remains unclear. Understanding this process will help describe the motility of N. gonorrheae bacteria, and thus the dissemination of the disease which they cause. In this article we track individual twitching cells and observe that their trajectories consist of alternating moving and pausing intervals, while the cell body is preferably oriented with its wide side toward the direction of motion. Based on these data, we propose a model for the collective pili operation of N. gonorrheae bacteria that explains the experimentally observed behavior. Individual pili function independently but can lead to coordinated motion or pausing via the force balance. The geometry of the cell defines its orientation during motion. We show that by changing pili substrate interactions, the motility pattern can be altered in a predictable way. Although the model proposed is tangibly simple, it still has sufficient robustness to incorporate further advanced pili features and various cell geometries to describe other bacteria that employ pili to move on surfaces.

Meningococcal resistance to antimicrobial peptides is mediated by bacterial adhesion and host cell RhoA and Cdc42 signalling.

Antimicrobial peptides (AMPs) constitute an essential part of the innate immune defence. Pathogenic bacteria have evolved numerous strategies to withstand AMP-mediated killing. The influence of host epithelia on bacterial AMP resistance is, however, still largely unknown. We found that adhesion to pharyngeal epithelial cells protected Neisseria meningitidis, a leading cause of meningitis and sepsis, from the human cathelicidin LL-37, the cationic model amphipathic peptide (MAP) and the peptaibol alamethicin, but not from polymyxin B. Adhesion to primary airway epithelia resulted in a similar increase in LL-37 resistance. The inhibition of selective host cell signalling mediated by RhoA and Cdc42 was found to abolish the adhesion-induced LL-37 resistance by a mechanism unrelated to the actin cytoskeleton. Moreover, N. meningitidis triggered the formation of cholesterol-rich membrane microdomains in pharyngeal epithelial cells, and host cell cholesterol proved to be essential for adhesion-induced resistance. Our data highlight the importance of Rho GTPase-dependent host cell signalling for meningococcal AMP resistance. These results indicate that N. meningitidis selectively exploits the epithelial microenvironment in order to protect itself from LL-37.

Identification of cell-penetrating peptides that are bactericidal to Neisseria meningitidis and prevent inflammatory responses upon infection.

Meningococcal disease is characterized by a fast progression and a high mortality rate. Cell-penetrating peptides (CPPs), developed as vectors for cargo delivery into eukaryotic cells, share structural features with antimicrobial peptides. A screen identified two CPPs, transportan-10 (TP10) and model amphipathic peptide (MAP), with bactericidal action against Neisseria meningitidis. Both peptides were active in human whole blood at micromolar concentrations, while hemolysis remained negligible. Additionally, TP10 exhibited significant antibacterial activity in vivo. Uptake of SYTOX green into live meningococci was observed within minutes after TP10 treatment, suggesting that TP10 may act by membrane permeabilization. Apart from its bactericidal activity, TP10 suppressed inflammatory cytokine release from macrophages infected with N. meningitidis as well as from macrophages stimulated with enterobacterial and meningococcal lipopolysaccharide (LPS). Finally, incubation with TP10 reduced the binding of LPS to macrophages. This novel endotoxin-inhibiting property of TP10, together with its antimicrobial activity in vivo, indicates the possibility to design peptide-based therapies for infectious diseases.

Loss of meningococcal PilU delays microcolony formation and attenuates virulence in vivo.

Neisseria meningitidis is a major cause of sepsis and bacterial meningitis worldwide. This bacterium expresses type IV pili (Tfp), which mediate important virulence traits such as the formation of bacterial aggregates, host cell adhesion, twitching motility, and DNA uptake. The meningococcal PilT protein is a hexameric ATPase that mediates pilus retraction. The PilU protein is produced from the pilT-pilU operon and shares a high degree of homology with PilT. The function of PilT in Tfp biology has been studied extensively, whereas the role of PilU remains poorly understood. Here we show that pilU mutants have delayed microcolony formation on host epithelial cells compared to the wild type, indicating that bacterium-bacterium interactions are affected. In normal human serum, the pilU mutant survived at a higher rate than that for wild-type bacteria. However, in a murine model of disease, mice infected with the pilT mutant demonstrated significantly reduced bacterial blood counts and survived at a higher rate than that for mice infected with the wild type. Infection of mice with the pilU mutant resulted in a trend of lower bacteremia, and still a significant increase in survival, than that of the wild type. In conclusion, these data suggest that PilU promotes timely microcolony formation and that both PilU and PilT are required for full bacterial virulence.

The complement regulator CD46 is bactericidal to Helicobacter pylori and blocks urease activity.

BACKGROUND & AIMS: CD46 is a C3b/C4b binding complement regulator and a receptor for several human pathogens. We examined the interaction between CD46 and Helicobacter pylori (a bacterium that colonizes the human gastric mucosa and causes gastritis), peptic ulcers, and cancer. METHODS: Using gastric epithelial cells, we analyzed a set of H pylori strains and mutants for their ability to interact with CD46 and/or influence CD46 expression. Bacterial interaction with full-length CD46 and small CD46 peptides was evaluated by flow cytometry, fluorescence microscopy, enzyme-linked immunosorbent assay, and bacterial survival analyses. RESULTS: H pylori infection caused shedding of CD46 into the extracellular environment. A soluble form of CD46 bound to H pylori and inhibited growth, in a dose- and time-dependent manner, by interacting with urease and alkyl hydroperoxide reductase, which are essential bacterial pathogenicity-associated factors. Binding of CD46 or CD46-derived synthetic peptides blocked the urease activity and ability of bacteria to survive in acidic environments. Oral administration of one CD46 peptide eradicated H pylori from infected mice. CONCLUSIONS: CD46 is an antimicrobial agent that can eradicate H pylori. CD46 peptides might be developed to treat H pylori infection.

Lactate causes downregulation of Helicobacter pylori adhesin genes sabA and labA while dampening the production of proinflammatory cytokines.

Chronic inflammation induced by Helicobacter pylori is strongly associated with gastric cancer development, which is influenced by both bacterial virulence and host genetics. The sialic acid-binding adhesin SabA and the MUC5AC-binding adhesin LabA are important H. pylori virulence factors that facilitate adhesion of the bacterium, which is a crucial step in colonization. Lactate utilization has been reported to play a key role in the pathogenicity of different bacterial species. However, this is poorly understood in H. pylori. In this study, we investigated the effect of lactate on H. pylori adhesin gene expression and the regulation of host inflammatory cytokines. We show that the bacterial adhesins SabA and LabA were downregulated at the transcriptional level during incubation of H. pylori with lactate. Downregulation of sabA required the involvement of the two-component system ArsRS, while labA was regulated via the CheA/CheY system, indicating differences in the regulation of these genes in response to lactate. The levels of the proinflammatory cytokines TNF and IL-6 in H. pylori-stimulated macrophages were reduced when lactate was present. Interestingly, glucose did not prevent the secretion of these cytokines. Taken together, our data suggest that lactate affects H. pylori adhesin gene expression and the host response upon infection.

Lactobacilli reduce cell cytotoxicity caused by Streptococcus pyogenes by producing lactic acid that degrades the toxic component lipoteichoic acid.

Lactobacilli are known to prevent colonization by many pathogens; nevertheless, the mechanisms of their protective effect are largely unknown. In this work, we investigated the role of lactobacilli during infection of epithelial cells with group A streptococci (GAS). GAS cause a variety of illnesses ranging from noninvasive disease to more severe invasive infections, such as necrotizing fasciitis and toxic shock-like syndrome. Invasion of deeper tissues is facilitated by GAS-induced apoptosis and cell death. We found that lactobacilli inhibit GAS-induced host cell cytotoxicity and shedding of the complement regulator CD46. Further, survival assays demonstrated that lactic acid secreted by lactobacilli is highly bactericidal toward GAS. In addition, lactic acid treatment of GAS, but not heat killing, prior to infection abolishes the cytotoxic effects against human cells. Since lipoteichoic acid (LTA) of GAS is heat resistant and cytotoxic, we explored the effects of lactic acid on LTA. By applying such an approach, we demonstrate that lactic acid reduces epithelial cell damage caused by GAS by degrading both secreted and cell-bound LTA. Taken together, our experiments reveal a mechanism by which lactobacilli prevent pathogen-induced host cell damage.

A regulatory role for macrophage class A scavenger receptors in TLR4-mediated LPS responses.

Recognition of microbial components by TLR, key sensors of infection, leads to induction of inflammatory responses. We found that, in vivo, TLR4 engagement by LPS induces up-regulation of the class A scavenger receptors (SR) macrophage receptor with a collagenous structure (MARCO) and SR-A, which occurs, at least in the case of MARCO, via both MyD88-dependent and -independent pathways. When challenging mice with a low dose of LPS followed by a high dose, class A SR-deficient mice showed a higher survival rate than WT mice. This was paired with increased production of IL-10 and anti-LPS Ab, as well as increased activation status of marginal zone B cells. However, the receptors were not crucial for survival when challenging mice i.p. with Neisseria meningitidis or Listeria monocytogenes, but they were found to contribute to microbial capture and clearance. This indicates physiological significance for the up-regulation of class A SR during early stages of bacterial infection. Thus, we believe that we have revealed a mechanism where SR regulate the activation status of the immune system and are involved in balancing a proper immune response to infection. This regulation could also be important in maintaining tolerance since these receptors have been shown to be involved in regulation of self-reactivity.

DNA Blocks the Lethal Effect of Human Beta-Defensin 2 Against Neisseria meningitidis.

Neisseria meningitidis is a gram-negative bacterium that often asymptomatically colonizes the human nasopharyngeal tract. These bacteria cross the epithelial barrier can cause life-threatening sepsis and/or meningitis. Antimicrobial peptides are one of the first lines of defense against invading bacterial pathogens. Human beta-defensin 2 (hBD2) is an antimicrobial peptide with broad antibacterial activity, although its mechanism of action is poorly understood. Here, we investigated the effect of hBD2 on N. meningitidis. We showed that hBD2 binds to and kills actively growing meningococcal cells. The lethal effect was evident after 2 h incubation with the peptide, which suggests a slow killing mechanism. Further, the membrane integrity was not changed during hBD2 treatment. Incubation with lethal doses of hBD2 decreased the presence of diplococci; the number and size of bacterial microcolonies/aggregates remained constant, indicating that planktonic bacteria may be more susceptible to the peptide. Meningococcal DNA bound hBD2 in mobility shift assays and inhibited the lethal effect of hBD2 in a dose-dependent manner both in suspension and biofilms, supporting the interaction between hBD2 and DNA. Taken together, the ability of meningococcal DNA to bind hBD2 opens the possibility that extracellular DNA due to bacterial lysis may be a means of N. meningitidis to evade immune defenses.

Endotoxin, capsule, and bacterial attachment contribute to Neisseria meningitidis resistance to the human antimicrobial peptide LL-37.

Pathogenic bacteria have evolved numerous mechanisms to evade the human immune system and have developed widespread resistance to traditional antibiotics. We studied the human pathogen Neisseria meningitidis and present evidence of novel mechanisms of resistance to the human antimicrobial peptide LL-37. We found that bacteria attached to host epithelial cells are resistant to 10 microM LL-37 whereas bacteria in solution or attached to plastic are killed, indicating that the cell microenvironment protects bacteria. The bacterial endotoxin lipooligosaccharide and the polysaccharide capsule contribute to LL-37 resistance, probably by preventing LL-37 from reaching the bacterial membrane, as more LL-37 reaches the bacterial membrane on both lipooligosaccharide-deficient and capsule-deficient mutants whereas both mutants are also more susceptible to LL-37 killing than the wild-type strain. N. meningitidis bacteria respond to sublethal doses of LL-37 and upregulate two of their capsule genes, siaC and siaD, which further results in upregulation of capsule biosynthesis.

Role of Sortase A in Lactobacillus gasseri Kx110A1 Adhesion to Gastric Epithelial Cells and Competitive Exclusion of Helicobacter pylori.

We have previously shown that Lactobacillus gasseri Kx110A1, a human stomach isolate, can colonize mouse stomach and reduce the initial colonization of Helicobacter pylori. Here, we investigated the role of sortase-dependent proteins (SDPs) involved in these functions by the construction of a mutant for srtA, the gene encoding the housekeeping sortase that covalently anchors SDPs to the cell surface. The srtA mutant showed a decrease in hydrophobicity and autoaggregation under acidic conditions, indicating the effect of SDPs on cell surface properties. Correspondingly, the srtA mutant lost the capacity to adhere to gastric epithelial cells, thus resulting in an inability to provide a physical barrier to prevent H. pylori adherence. These results indicate that sortase A is a key determinant of the cell surface properties of L. gasseri Kx110A1 and contributes to Lactobacillus-mediated exclusion of H. pylori. Understanding the molecular mechanisms by which lactobacilli antagonize H. pylori might contribute to the development of novel therapeutic strategies that take advantage of health-promoting bacteria and reduce the burden of antibiotic resistance.

Deletion of D-Lactate Dehydrogenase A in Neisseria meningitidis Promotes Biofilm Formation Through Increased Autolysis and Extracellular DNA Release.

Neisseria meningitidis is a Gram-negative bacterium that asymptomatically colonizes the human nasopharyngeal mucosa. Pilus-mediated initial adherence of N. meningitidis to the epithelial mucosa is followed by the formation of three-dimensional aggregates, called microcolonies. Dispersal from microcolonies contributes to the transmission of N. meningitidis across the epithelial mucosa. We have recently discovered that environmental concentrations of host cell-derived lactate influences N. meningitidis microcolony dispersal. Here, we examined the ability of N. meningitidis mutants deficient in lactate metabolism to form biofilms. A lactate dehydrogenease A (ldhA) mutant had an increased level of biofilm formation. Deletion of ldhA increased the N. meningitidis cell surface hydrophobicity and aggregation. In this study, we used FAM20, which belongs to clonal complex ST-11 that forms biofilms independently of extracellular DNA (eDNA). However, treatment with DNase I abolished the increased biofilm formation and aggregation of the ldhA-deficient mutant, suggesting a critical role for eDNA. Compared to wild-type, the ldhA-deficient mutant exhibited an increased autolytic rate, with significant increases in the eDNA concentrations in the culture supernatants and in biofilms. Within the ldhA mutant biofilm, the transcription levels of the capsule, pilus, and bacterial lysis genes were downregulated, while norB, which is associated with anaerobic respiration, was upregulated. These findings suggest that the absence of ldhA in N. meningitidis promotes biofilm formation and aggregation through autolysis-mediated DNA release.