The Role of luxS in Histophilus somni Virulence and Biofilm Formation

Abstract

S-Ribosylhomocysteinase (LuxS) is required for the synthesis of the autoinducer-2 (AI-2) quorum-sensing signaling molecule in many Gram-negative bacteria. The bovine (and ovine) opportunistic pathogen Histophilus somni contains luxS and forms a biofilm containing an exopolysaccharide (EPS) in the matrix. Since biofilm formation is regulated by quorum sensing in many bacteria, the roles of luxS in H. somni virulence and biofilm formation were investigated. Although culture supernatants from H. somni were ineffective at inducing bioluminescence in the Vibrio harveyi reporter strain BB170, H. somniluxS complemented the biosynthesis of AI-2 in the luxS-deficient Escherichia coli strain DH5α. H. somni strain 2336 luxS was inactivated by transposon mutagenesis. RNA expression profiles revealed that many genes were significantly differentially expressed in the luxS mutant compared to that in the wild-type, whether the bacteria were grown planktonically or in a biofilm. Furthermore, the luxS mutant had a truncated and asialylated lipooligosaccharide (LOS) and was substantially more serum sensitive than the wild-type. Not surprisingly, the luxS mutant was attenuated in a mouse model for H. somni virulence, and some of the altered phenotypes were partially restored after the mutation was complemented with a functional luxS However, no major differences were observed between the wild-type and the luxS mutant in regard to outer membrane protein profiles, biofilm formation, EPS production, or intracellular survival. These results indicate that luxS plays a role in H. somni virulence in the context of LOS biosynthesis but not biofilm formation or other phenotypic properties examined.

Keywords: Histophilus somni; RNA-sequencing; biofilm; lipooligosaccharide; luxS; mouse model; mutagenesis; virulence.

FIG 1

Luminescence production by V. harveyi reporter strain BB170 grown in the presence of culture supernatants from different bacteria: VHBB152, V. harveyi strain BB152 (positive control); ABM, autoinducer bioassay medium (negative control); ECLUXS, E. coli DH5α containing pCR2.1TOPOluxS; ECTOPO, E. coli DH5α carrying the TOPO vector only (negative control); HFLU, Haemophilus influenzae; GPS, Glaesserella parasuis; APP, Actinobacillus pleuropneumoniae; MHA1, Mannheimia haemolytica A1; HS, Histophilus somni.

FIG 2

Comparison of transcription levels of genes in the wild-type and the luxS mutant cultured planktonically. Red dots represent genes whose transcription levels were altered significantly in the luxS mutant compared to that in the wild-type. Gray dots represent genes whose transcription levels were not affected significantly. The dots above the diagonal line represent genes that were upregulated, while the dots below the diagonal line represent genes that were downregulated. Generally, the number of genes downregulated was higher in the luxS mutant than in the wild-type. The differences in transcription levels were expressed as log ratio. Log₂ (fold change) of greater or equal to 1 or less than or equal to −1 was considered significant. Specific genes are shown in Table S1 in the supplemental material.

FIG 3

Venn diagram showing number of genes differentially expressed when bacteria grown as a biofilm were compared to those that were grown planktonically. Genes (n = 359) up- or downregulated in both the wild-type and luxS mutant strains are shown in the center. Genes (n = 1,142) differentially regulated only in the wild-type strain are shown on the left. Genes (n = 1,226) differentially regulated only in the luxS mutant are shown on the right. Locus tags and log₂ (fold change) of these genes are shown in Tables S2, S3, and S4.

FIG 4

Comparison of LOS electrophoretic profiles and serum susceptibility of strains 2336, 2336::TnluxS, and complemented mutants of 2336::TnluxS. (A) Odd-numbered lanes, LOS extracted from H. somni grown in broth lacking sialic acid; even-numbered lanes, LOS from bacteria grown with 100 μg/ml sialic acid. Lanes 1 and 2, strain 2336; lanes 3 and 4, 2336::TnluxS; lanes 5 and 6, 2336::TnluxS^CT; lanes 7 and 8, 2336::TnluxS^CV; lanes 9 and 10, 2336::TnluxS^CP; lanes 11 and 12, 2336::TnluxS^CQ. The upper bands were partially restored in the LOS electrophoretic profiles of the complemented mutants and were more visible with the addition of sialic acid, supporting the presence of the restored chain and terminal galactose. (B) The data are shown as percent survival. Strain 2336 was resistant to serum killing at concentrations of up to 50%, while mutant 2336::TnluxS was sensitive at these concentrations. The serum resistance of the complemented luxS mutants was partially restored.

FIG 5

Growth and confocal microscopy of H. somni strains in bovine monocytes (BM). (A) H. somni strains 2336, 2336::TnluxS, and 129Pt were incubated with BM for up to 72 h. Strain 129Pt is an avirulent, serum-sensitive control strain. The luxS mutant strain diminished in numbers in BM during the first 24 h of incubation, whereas avirulent strain 129Pt was readily killed within 24 h. However, after 24 h, the growth rates were similar between the luxS mutant and the wild-type within the BM. (B) Confocal microscopy of H. somni wild-type and luxS mutant in macrophages. The red arrows indicate the locations of the marker EEA-1 or LAMP-2; the green arrows indicate the bacteria; the yellow arrows indicate the colocalization of the markers and the bacteria. Both the wild-type and luxS mutant colocalized with EEA-1, but not LAMP-2, and did not acidify phagosomes. The numbers of intracellular bacteria of both the wild-type and luxS mutant were also similar. Strain 129Pt was previously shown to colocalize with both EEA-1 and LAMP-2 and to acidify phagolysosomes (33).

FIG 6

Comparison of wild-type and mutant H. somni biofilms. (A) Scanning electron microscopy of biofilms formed by the wild-type (W) and luxS mutant (M). The biofilms formed by the two strains appeared similar, indicating that the luxS mutation had little, if any, effect on biofilm formation. (B) Biofilms formed by the wild-type, luxS mutant, and luxS-complemented strains of H. somni were quantified using crystal violet staining. Differences in the amount of biofilm formed between all strains were not significantly different (P > 0.05).