Neisseria meningitidis (Nm) and Neisseria lactamica (Nl) are commensal bacteria that live in the human nasopharynx, where they form microcolonies. In contrast to Nl, Nm occasionally causes blood and/or meningitis infection with often fatal consequences. Here, we studied interactions between neisserial strains during biofilm formation. Fluorescent strains were engineered and analyzed for growth in single- and dual-strain biofilms with confocal laser-scanning microscopy. Different strains of diverse Neisseria species formed microcolonies of different sizes and morphologies. Pair-wise combinations of two invasive Nm strains and one Nm carrier isolate showed that these strains can coexist in spite of the fact that they produce toxins to combat congeners. This lack of competition was even observed when the biofilms were formed under nutrient limitation and can be explained by the observation that the separate microcolonies within mixed biofilms are mostly lineage specific. However, these microcolonies showed different levels of interaction. The coexistence of two strains was also observed in mixed biofilms of Nm and Nl strains. Inactivation of the autotransporter NalP, which prevents the release of the heparin-binding antigen NHBA and the α-peptide of IgA protease from the cell surface, and/or the production of autotransporter AutA increased interactions between microcolonies, as evidenced by close contacts between microcolonies on the substratum. Qualitative and quantitative analysis revealed an altered spatial distribution of each strain in mixed biofilms with consequences for the biomass, biofilm architecture and bacterial viability depending on the synthesis of NalP and AutA, the expression of which is prone to phase variation. Being in a consortium resulted in some cases in commensalism and cooperative behavior, which promoted attachment to the substratum or increased survival, possibly as result of the shared use of the biofilm matrix. We hypothesize that Nm strains can cooperate during host colonization, but, possibly, the different capacities of the microcolonies of each strain to resist the host's defenses limits the long-term coexistence of strains in the host.

Summary Yersinia ruckeri causes enteric redmouth disease (ERM) that mainly affects salmonid fishes and leads to significant economic losses in the aquaculture industry. An increasing number of outbreaks and the lack of effective vaccines against some serotypes necessitates novel measures to control ERM. Importantly, Y. ruckeri survives in the environment for long periods, presumably by forming biofilms. How the pathogen forms biofilms and which molecular factors are involved in this process, remains unclear. Yersinia ruckeri produces two surface‐exposed adhesins, belonging to the inverse autotransporters (IATs), called Y. ruckeri invasin (YrInv) and Y. ruckeri invasin‐like molecule (YrIlm). Here, we investigated whether YrInv and YrIlm play a role in biofilm formation and virulence. Functional assays revealed that YrInv and YrIlm promote biofilm formation on different abiotic substrates. Confocal microscopy revealed that they are involved in microcolony interaction and formation, respectively. The effect of both IATs on biofilm formation correlated with the presence of different biopolymers in the biofilm matrix, including extracellular DNA, RNA and proteins. Moreover, YrInv and YrIlm contributed to virulence in the Galleria mellonella infection model. Taken together, we propose that both IATs are possible targets for the development of novel diagnostic and preventative strategies to control ERM.

Lipopolysaccharides are anchored to the outer membrane of Gram-negative bacteria by a hydrophobic moiety known as lipid A, which potently activates the host innate immune response. Lipid A of Bordetella pertussis, the causative agent of whooping cough, displays unusual structural asymmetry with respect to the length of the acyl chains at the 3 and 3′ positions, which are 3OH-C10 and 3OH-C14 chains, respectively. Both chains are attached by the acyltransferase LpxA, the first enzyme in the lipid A biosynthesis pathway, which, in B. pertussis, has limited chain length specificity. However, this only partially explains the strict asymmetry of lipid A. In attempts to modulate the endotoxicity of B. pertussis lipid A, here we expressed the gene encoding LpxA from Neisseria meningitidis, which specifically attaches 3OH-C12 chains, in B. pertussis. This expression was lethal, suggesting that one of the downstream enzymes in the lipid A biosynthesis pathway in B. pertussis cannot handle precursors with a 3OH-C12 chain. We considered that the UDP-diacylglucosamine pyrophosphohydrolase LpxH could be responsible for this defect as well as for the asymmetry of B. pertussis lipid A. Expression of meningococcal LpxH in B. pertussis indeed resulted in new symmetric lipid A species with 3OH-C10 or 3OH-C14 chains at both the 3 and 3′ positions, as revealed by MS analysis. Furthermore, co-expression of meningococcal lpxH and lpxA resulted in viable cells that incorporated 3OH-C12 chains in B. pertussis lipid A. We conclude that the asymmetry of B. pertussis lipid A is determined by the acyl chain length specificity of LpxH.

Whole-cell vaccines against Gram-negative bacteria commonly display high reactogenicity caused by the endotoxic activity of lipopolysaccharide (LPS), one of the major components of the bacterial outer membrane. Underacylation of the lipid A moiety of LPS has been related with reduced endotoxicity in several Gram-negative species. Here, we evaluated whether the inactivation of two genes encoding lipid A acylases of Bordetella bronchiseptica, i.e. pagP and lpxL1, could be used for the development of less reactogenic vaccines against this pathogen for livestock and companion animals. Inactivation of pagP resulted in the loss of the secondary palmitate chain at position 3' of lipid A, but hardly affected the potency of the LPS to activate the Toll-like receptor 4 (TLR4). Inactivation of lpxL1 resulted in the loss of the secondary 2-hydroxy laurate group present at position 2 of lipid A and, unexpectedly, in the additional loss of the glucosamines that decorate the phosphate groups at positions 1 and 4' and in an increase in LPS molecules carrying O-antigen. The resulting LPS showed greatly reduced potency to activate TLR4 in HEK-Blue reporter cells expressing human or mouse TLR4 as well as in porcine macrophages. Characterization of the lpxL1 mutant revealed many pleiotropic phenotypes, including increased resistance to SDS and rifampicin, increased susceptibility to cationic antimicrobial peptides, decreased auto-aggregation and biofilm formation, and a tendency to decreased infectivity of macrophages, which are all related to the altered LPS structure. We suggest that the lpxL1 mutant will be useful for the generation of safer vaccines.

The Gram-negative bacterium Bordetella pertussis is the causative agent of a respiratory infection known as whooping cough. Previously developed whole-cell pertussis vaccines were effective, but appeared to be too reactogenic mainly due to the presence of lipopolysaccharide (LPS, also known as endotoxin) in the outer membrane (OM). Here, we investigated the possibility of reducing endotoxicity by modulating the LPS levels. The promoter of the lpxC gene, which encodes the first committed enzyme in LPS biosynthesis, was replaced by an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter. The IPTG was essential for growth, even when the construct was moved into a strain that should allow for the replacement of LPS in the outer leaflet of the OM with phospholipids by defective phospholipid transporter Mla and OM phospholipase A. LpxC depletion in the absence of IPTG resulted in morphological changes of the cells and in overproduction of outer-membrane vesicles (OMVs). The reduced amounts of LPS in whole-cell preparations and in isolated OMVs of LpxC-depleted cells resulted in lower activation of Toll-like receptor 4 in HEK-Blue reporter cells. We suggest that, besides lipid A engineering, also a reduction in LPS synthesis is an attractive strategy for the production of either whole-cell- or OMV-based vaccines, with reduced reactogenicity for B. pertussis and other Gram-negative bacteria.