Histophilus somni Survives in Bovine Macrophages by Interfering with Phagosome-Lysosome Fusion but Requires IbpA for Optimal Serum Resistance

Abstract

Histophilus somni is capable of intracellular survival within professional phagocytic cells, but the mechanism of survival is not understood. The Fic motif within the direct repeat (DR1)/DR2 domains of the IbpA fibrillary network protein of H. somni is cytotoxic to epithelial and phagocytic cells, which may interfere with the bactericidal activity of these cells. To determine the contribution of IbpA and Fic to resistance to host defenses, H. somni strains and mutants that lacked all or a region of ibpA (including the DR1/DR2 regions) were tested for survival in bovine monocytic cells and for serum susceptibility. An H. somni mutant lacking IbpA, but not the DR1/DR2 region within ibpA, was more susceptible to killing by antiserum than the parent, indicating that the entire protein was associated with serum resistance. H. somni strains expressing IbpA replicated in bovine monocytes for at least 72 h and were toxic for these cells. Virulent strain 2336 mutants lacking the entire ibpA gene or both DR1 and DR2 were not toxic to the monocytes but still survived within the monocytes for at least 72 h. Monitoring of intracellular trafficking of H. somni with monoclonal antibodies to phagosomal markers indicated that the early phagosomal marker early endosome antigen 1 colocalized with all isolates tested, but only strains that could survive intracellularly did not colocalize with the late lysosomal marker lysosome-associated membrane protein 2 and prevented the acidification of phagosomes. These results indicated that virulent isolates of H. somni were capable of surviving within phagocytic cells through interference in phagosome-lysosome maturation. Therefore, H. somni may be considered a permissive intracellular pathogen.

Keywords: Fic motif; Histophilus somni; IbpA; bactericidal activity; intracellular bacteria; monocytes; phagocytosis.

FIG 1

Survival of H. somni strains 2336 and 129Pt in BPBM and BM cell lines. The bacteria were incubated with monocytes for 1 h (uptake time) at a multiplicity of infection of 100:1 (bacteria to monocytes); gentamicin was added to kill extracellular bacteria; after 30 min, the monocytes were washed, incubated for 0, 24, 48, or 72 h, and lysed; and the released bacteria were cultured on blood agar. Results represent the means ± standard deviations from at least 3 experiments. Standard deviation bars are difficult to see due to their small size and the symbols.

FIG 2

Survival of H. somni strains in BM cells after 24 h of coincubation. Percent survival of H. somni was determined by lysis of BM cells after 1 h (incubation time given for uptake) and 24 h after coincubation and culture. The number of colonies recovered after 24 h of incubation was divided by the number of colonies after 1 h of incubation and multiplied by 100. Results represent the means ± standard deviations from 3 experiments.

FIG 3

Percent intracellular survival of H. somni strain 129Pt bacteria after 24 h of incubation in BM cells preincubated for 2 h with or without 20-fold- or 4-fold-concentrated, semipurified IbpA from the strain 2336 supernatant. The survival of strain 2336 in BM cells is shown for comparison. Results represent the means ± standard deviations from at least 3 experiments.

FIG 4

Percent survival of H. somni preputial isolates after 24 h of incubation in BM cells that were preincubated for 2 h with a 20-fold-concentrated culture supernatant containing IbpA. Those strains that lacked IbpA were significantly more resistant to intracellular killing by BM monocytes after the addition of IbpA (strains 1P, 133P, 130Pf, and 129Pt). Strains 24P and 124P, and 20P, produced IbpA, and there was little, or no, difference, respectively, in killing by BM cells following preincubation of the cells with IbpA. Results represent the means ± standard deviations from at least 3 experiments.

FIG 5

Intracellular survival of H. somni ibpA mutants and control strains in BM cells over 72 h. The lack of DR1/DR2 regions containing the cytotoxic Fic motifs (2336ΔIbpA9) or the lack of the entire IbpA protein (2336ΔIbpA1) had no significant effect on the intracellular survival of strain 2336 in BM cells. Results represent the means ± standard deviations from at least 3 experiments.

FIG 6

Confocal microscopy of H. somni strain 2336, strain 129Pt with and without the addition of IbpA, and strain 64Vc following phagocytosis. The markers EEA-1 (10 min), LAMP-2 (3 h), and LysoTracker (3 h) were stained with Alexa Fluor 546 (red), and H. somni was stained with Alexa Fluor 488 (green). The red arrowheads point to the markers, the green arrowheads point to H. somni, and the yellow arrowheads point to colocalization of the markers with H. somni. Each photo shown is a representative field of 6 to 10 fields examined from 3 separate experiments.

FIG 7

Quantification of the number of bacteria of H. somni serum-resistant strains 2336 and 64Vc and serum-sensitive strain 129Pt (with and without added IbpA) that colocalized with the marker EEA-1, LysoTracker, or LAMP-2. The level of colocalization of H. somni bacteria with each marker was quantified at either 10 min (EEA-1) or 3 h (LysoTracker/acidification and LAMP-2). Quantification of the levels of the EEA-1 and LAMP-2 markers, and LysoTracker, was performed by using ImageJ and Oufti software, which analyze the percentage of fluorescent dots and fluorescence signals, respectively. Results represent the means ± standard deviations from at least 3 experiments.

FIG 8

Serum resistance of H. somni strain 2336 and its ibpA mutants. Mutant and control strains were tested for susceptibility to killing by antiserum to H. somni lipooligosaccharide and bovine complement. Shown are data for control parent strain 2336 (serum resistant), mutant 2336ΔIbpA1 lacking the entire ibpA gene, mutant 2336ΔIbpA9 lacking only DR1/DR2 regions containing the toxic Fic motifs, and control strain 129Pt (serum sensitive). Results represent the means ± standard deviations from at least 3 experiments.