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

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.

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.

INTRODUCTION

The Gram-negative bacterium Histophilus somni is an opportunistic pathogen associated with bovine respiratory disease and multisystemic diseases in cattle and sometimes sheep, including thrombotic meningoencephalitis (TME), myocarditis, arthritis, mastitis, reproductive failure and abortion, and others, probably resulting from bacteremia (1). However, some strains of H. somni are serum sensitive, and at least one such strain (129Pt) lacks many of the virulence factors associated with disease isolates (2). The only known reservoirs for H. somni are the mucosal sites of ruminants (3).

Virulent strains of H. somni possess a wide variety of physiological properties and mechanisms that enable the bacteria to resist the bactericidal effects of host defenses or to modulate host immune cells. Such mechanisms include phase variation of lipooligosaccharide (LOS), modification of LOS with sialic acid and phosphorylcholine (4), apoptosis of endothelial cells and neutrophils with disruption of intercellular junctions (5), and biofilm formation (6). Furthermore, the bacteria secrete a fibrillar and surface-associated immunoglobulin binding protein (IbpA), the N-terminal region of which is capable of binding immunoglobulins through their Fc component and may also contribute to the adherence of the bacteria to host cells (7). The COOH terminus of IbpA has homology to a region in Yersinia species YopT but lacks cytotoxic activity (8). In contrast, sequence analysis of ibpA indicates that there are two direct repeats (DR1 and DR2) just upstream of the yopT-like region, each containing a filamentation-induced-by-cAMP (Fic) motif, HPFxxGNGR (8). These Fic domains can be found in both bacterial and eukaryotic cells. In H. somni, the Fic motifs of both DR1 and DR2 have been shown to be toxic for bovine epithelial and phagocytic cells, resulting in rounding of the cells, increased detachment of infected macrophages, and disruption of actin fibers (9, 10). H. somni strain 2336 can inhibit phagocytosis of microspheres by primary bovine monocytes (BMs), but a mutant with essentially the entire ibpA gene deleted cannot (10). Antibodies to the recombinant DR2 region of IbpA can neutralize the cytotoxic effect on these cells (11). Immunization of mice and calves with recombinant DR2 also protects the animals from H. somni bacteremia and pneumonia, respectively (12, 13). The presence of IbpA in H. somni strains is also associated with serum resistance (7).

Virulent strains of H. somni are capable of surviving within bovine polymorphonuclear leukocytes (PMNs), monocytes, and macrophages (14, 15). Phagocytic cells infected with live H. somni bacteria are less capable of internalizing a secondary target, such as opsonized Staphylococcus aureus and microspheres (16, 17). Killed, whole bacteria or supernatants from heat-killed bacteria can also inhibit the internalization of S. aureus by PMNs but not bovine macrophages (16, 17). We have previously reported that the oxidative burst generated by phagocytic cells in contact with viable disease isolates of H. somni is significantly inhibited. However, there is no inhibition of the oxidative burst by killed H. somni bacteria, nonvirulent mucosal strain 129Pt, and heterologous strains, which include Haemophilus influenzae and Brucella abortus (18). The mechanism by which H. somni survives within phagocytic cells remains unclear. Because the Fic motifs within IbpA are toxic to phagocytic cells and induce disruption of actin filaments, it is possible that H. somni survives intracellular killing through Fic-mediated interference of phagocytotic cell functions. In this study, we used various mutants with transposon (Tn) insertions and in-frame deletions in ibpA to determine the contribution of IbpA and the Fic motifs to serum susceptibility and intracellular killing of H. somni and how virulent disease isolates and avirulent isolates traffic within bovine monocytes.

RESULTS

Intracellular survival of H. somni in bovine monocyte and bovine peripheral blood monocyte cells.

The ability of H. somni strains 2336 and 129Pt to survive intracellularly in bovine monocyte (BM), bovine FBM-17, mouse J774A.1, and human THP cells was examined in comparison to freshly collected bovine peripheral blood monocyte (BPBM) cells. H. somni pathogenic strain 2336 survived in BPBMs and was cytotoxic to these cells, resulting in detachment and rounding of the cells (data not shown), as previously described for FBM-17 cells (9). Strain 2336 also grew well in the BM cell line. Although the inoculum of strain 2336 cells in BPBMs was more than 1 log higher than that in BM cells, the slopes of the growth curve lines over 72 h for strain 2336 in BPBM and BM cells were almost the same (0.022 and 0.026, respectively). In contrast, mucosal strain 129Pt from the healthy prepuce was not cytotoxic and did not survive in BPBM or BM cells (Fig. 1). The mouse macrophage cell line J774.1, bovine FBM-17 cells, and human leukemic THP-1 cells were also tested but were unable to kill strain 129Pt (data not shown). Therefore, BM cells were used for all subsequent studies.

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.

Several H. somni strains from disease sites and healthy mucosal sites were tested for survival within BM cells following phagocytosis. The presence or absence of ibpA, which contains the cytotoxic Fic domains within DR1/DR2 (9), in all the H. somni strains used in this study has previously been described (Table 1) (19). All the disease isolates and most of the vaginal isolates tested were capable of intracellular replication in BM cells, but most preputial isolates tested were not (Table 1 and Fig. 2). Strains 1P, 129Pt, 130Pf, and 133P from the bovine prepuce do not produce IbpA (19) and were unable to replicate intracellularly after 24 h of coincubation (Table 1 and Fig. 2). However, some preputial isolates previously shown to produce IbpA (24P, 124P, and 20P) were also killed by BM cells, although preputial isolate 22P, which produces IbpA, was resistant. Of interest was that strain 1225, which was isolated from the bovine prepuce in The Netherlands, was highly resistant to intracellular killing, but it was unknown whether this isolate was associated with disease or expressed IbpA. Therefore, the expression of IbpA was not universally associated with intracellular survival.

TABLE 1.

H. somni strains used in this study

Strain	Description	Presence of IbpAa	Serum resistance	Reference(s) or source
2336	Bovine pneumonia strain	+	+	26
2336::TnfhaB mutant 3b	ibpA Tn mutant		NDc	29
2336::TnfhaB mutant 13	ibpA Tn mutant		ND	29
2336::TnfhaB mutant 91	ibpA Tn mutant		ND	29
2336::TnfhaB mutant 9	ibpA Tn mutant		ND	29
2336::TnfhaB mutant 27	ibpA Tn mutant		ND	29
2336::TnfhaB mutant 23	ibpA Tn mutant		ND	29
2336::TnfhaB mutant 137	ibpA Tn mutant		ND	29
2336ΔibpA1	ibpA deletion mutant (entire ibpA gene)		−	9
2336ΔibpA5	ibpA deletion mutant (3′-terminal sequence)		ND	38, 39
2336ΔibpA7	ibpA deletion mutant (DR2 sequence)		ND	38, 39
2336ΔibpA8	ibpA deletion mutant (DR1 sequence)		ND	38, 39
2336ΔibpA9	ibpA deletion mutant (DR1 and DR2 sequences and the entire Fic motif)		+	38, 39
2336ΔibpA11	ibpA deletion mutant (R1R2 sequence)		ND	38, 39
129Pt	Normal prepuce	−	−	26
1P	Normal prepuce	−	−	26
130Pf	Normal prepuce	−	−	26
133P	Normal prepuce	−	−	26
124P	Normal prepuce	+	−	26
20P	Normal prepuce	+	+	26
24P	Normal prepuce	+	−	26
22P	Normal prepuce	+	+	26
1225	Prepuce	ND	ND	V. Fussing, National Veterinary Laboratory, Denmark
221V	Normal vagina	+	−	26
80	Normal vagina	+	+	26
29Vb	Normal vagina	+	−	26
41Vc	Normal vagina	+	−	26
208V	Normal vagina	+	−	26
202V	Normal vagina	+	−	26
64Vc	Normal vagina	+	+	26
1297	Pneumonia	+	+	26
5166	Pneumonia	ND	ND	26
738	Phase variant of strain 2336 isolated from challenged calf lung	+	+	47
318	Phase variant of strain 2336	+	+	26
8025	TME	+	+	26
2089	Abortion	+	+	26
649	Abortion	+	+	26
570	Abortion	+	−	26

^aThe presence or absence of ibpA in wild-type strains was determined by PCR, and the production of IbpA was determined by Western blotting of the bacterial culture supernatant (19).

^bAll mutants were derived from strain 2336.

^cND, not determined.

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.

Role of IbpA in intracellular survival of H. somni.

To assess the direct effect of IbpA on H. somni intracellular survival, BM cells were incubated for 2 h with an H. somni culture supernatant that was concentrated 1:4 or 1:20 to enrich for IbpA and then infected with H. somni strain 129Pt, which cannot survive intracellularly. After 24 h of incubation with the concentrated culture supernatant, the intracellular survival of strain 129Pt in BM cells was significantly (P < 0.001) greater than that for the bacteria within BM cells not incubated with IbpA, and this effect was dose dependent (Fig. 3). These results supported the hypothesis that IbpA had an adverse effect on BM cells, inhibiting their ability to subsequently take up and/or kill strain 129Pt bacteria. IbpA (20-fold concentrate) also significantly enhanced the intracellular survival of the other H. somni strains (24 h of incubation in monocytes) that were not as susceptible to intracellular killing as strain 129Pt but also lacked ibpA (1P, 133P, and 130Pf [P = 0.004, 0.001, and 0.005, respectively]). However, the enhancement of survival for these strains was not as dramatic as for strain 129Pt (Fig. 4). Furthermore, the addition of IbpA only moderately enhanced the intracellular survival of strain 24P and had no effect on strains 124P and 20P (Fig. 4), all of which, as expected, produce IbpA (19).

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.

Survival of H. somni ibpA mutants in BM cells.

Several mutants with Tn insertion mutations in ibpA were selected from a bank of Tn mutants for intracellular survival in BM cells. All the mutants replicated significantly more slowly than the parent strain at 24 h postincubation within BM cells (P < 0.001), but none of the mutants demonstrated a significant difference in the number of viable intracellular bacteria after 48 or 72 h of incubation (P > 0.05) (data not shown). However, in all of these mutants, the Tn had inserted at the C-terminal region that is responsible for Fc binding by IbpA, and none of these mutants contained the transposon within the DR1/DR2 region. Therefore, mutants with the entire ibpA gene replaced with a kanamycin resistance (Kn^r) gene or with in-frame deletions in ibpA were also tested for intracellular survival (Fig. 5). Mutants with essentially the entire ibpA gene removed (2336ΔIbpA1) or only the region containing both DR1 and DR2 (DR1/DR2) and the Fic motifs (2336ΔIbpA9) were capable of surviving within BM cells as effectively as parent strain 2336. Mutants 2336ΔIbpA5 (deletion near the 3′ terminus), 2336ΔIbpA7 (only DR2 deleted), 2336ΔIbpA8 (only DR1 deleted), and 2336ΔIbpA11 (deletion of R1R2 sequences) were also no more susceptible to intracellular killing than the parent (data not shown). Therefore, destruction of actin filaments and cell toxicity due to the Fic motifs were not, by themselves, the mechanism by which H. somni strain 2336 survived within phagocytic cells. Furthermore, there was not a significant difference between the presence or absence of IbpA and DR1/DR2 and the uptake of the bacteria.

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.

Intracellular trafficking of H. somni within BM cells.

Intracellular trafficking of pneumonia isolate strain 2336 and mucosal isolate strain 129Pt was examined by confocal microscopy to further clarify the mechanism of H. somni intracellular survival. The early phagosomal marker early endosome antigen 1 (EEA-1) was expressed in BM cells and colocalized with strain 129Pt, with and without the addition of IbpA, and with strain 2336. Phagolysosomes containing strain 129Pt with and without the addition of IbpA were also acidified and colocalized with lysosome-associated membrane protein 2 (LAMP-2) (Fig. 6 and 7) (P > 0.05). Therefore, intracellular trafficking of strain 129Pt in BM cells was not affected by the addition of IbpA. However, although the colocalization of EEA-1 with strain 2336 was similar to that of strain 129Pt, the levels of acidification of phagolysosomes and expression/colocalization of LAMP-2 in BM cells infected with strain 2336 were significantly lower than with strain 129Pt (Fig. 6 and 7) (P = 0.008).

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.

The vaginal isolates used in this study were also capable of surviving in BM cells, although these isolates were not associated with disease. Therefore, the intracellular trafficking of strain 64Vc, a typical vaginal isolate, was also examined by confocal microscopy. Strain 64Vc colocalized effectively with the early phagosomal marker EEA-1 10 min after infection. However, colocalization of LAMP-2 with strain 64Vc and the acidification of the phagosomes were, as described above for strain 2336, significantly reduced compared to the same measurements with strain 129Pt (P = 0.01 and P = 0.001, respectively) (Fig. 6 and 7).

Serum susceptibility of IbpA and DR1/DR2 mutants.

The presence of IbpA in H. somni is also associated with serum resistance (7). Therefore, we sought to determine if a serum-resistant strain shifts to serum sensitivity with the loss of the entire IbpA protein or only the DR1/DR2 regions. Serum resistance is relative in H. somni; even “serum-resistant” strains can be killed in the presence of adequate antibodies to H. somni surface antigens. Therefore, to maximize the serum bactericidal effect, antiserum to H. somni LOS was used in the presence of precolostrum calf serum (an antibody-free source of complement). In the absence of any antiserum, the numbers of bacteria of strain 2336 and mutants lacking essentially the entire IbpA protein (H. somni 2336ΔIbpA1) or both DR1 and DR2 (H. somni 26336ΔIbpA9) were increased, indicating that these bacteria were resistant to the effects of complement alone (Fig. 8). When anti-LOS serum at a final concentration of 40% (vol/vol) was added, all the strains were effectively killed. However, when anti-LOS serum was added at a final concentration of between 10% and 30% (vol/vol), H. somni mutant 2336ΔIbpA9, lacking DR1/DR2, was significantly more resistant to killing than even strain 2336 (P < 0.008), but H. somni mutant 2336ΔIbpA1 (lacking the entire IbpA protein) was more susceptible to killing in 10% antiserum (P = 0.004) than strain 2336, although it was not as susceptible as strain 129Pt. Therefore, the IbpA protein, but not DR1 and DR2 containing the Fic motifs, contributed to serum resistance. However, other factors that contribute to serum resistance also appear to be deficient in H. somni strain 129Pt.

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.

DISCUSSION

Virulent strains of H. somni are readily phagocytosed, but not killed, by neutrophils, macrophages, or monocytic cells (14, 15, 20). However, the mechanism by which H. somni survives within these cells is not clear. Generation of reactive oxygen intermediates is an important defense mechanism that phagocytic cells use to kill bacteria following phagocytosis (21). Inhibition of the oxidative burst by phagocytic cells following incubation with disease isolates of H. somni, but not serum-sensitive isolates from the normal bovine prepuce, has been well established and may contribute to intracellular survival (14, 17, 18, 22–24). In addition, incubation of phagocytic cells with H. somni inhibits their subsequent uptake of opsonized S. aureus (16, 17, 22), indicating that the cells have been compromised in regard to phagocytic capacity following incubation with H. somni. Several investigators have shown that inhibition of the oxidative burst requires contact with, or the presence of, viable H. somni bacteria (16, 18, 22, 24), whereas others have reported that this inhibitory activity can occur by killed cells or cell fractions (17, 25). The variation in these results may be related to differences in the assays used.

BM cells killed strain 129Pt as effectively as BPBM cells, but bovine FBM-17 cells, murine J774A.1 cells, and human THP cells did not. Strain 2336 survived within BM cells as well as within BPBMs, indicating that this primary cell line was a suitable BPBM surrogate for use in these assays and for studies involving the interaction of H. somni with bovine phagocytic cells. Many additional disease and mucosal isolates were also tested for intracellular survival. However, while most preputial isolates from healthy animals were confirmed to be less capable of replicating in monocytes, isolates from the healthy vagina were as capable of surviving intracellularly as disease isolates. Of interest is that the isolates resistant to intracellular killing are also serum resistant (19, 26).

The production of extracellular protein toxins by H. somni has not been reported. However, high-molecular-weight fibrillar proteins that bind IgG2 are present on the cell surface of all H. somni strains tested, except for some preputial isolates (19, 27, 28). This high-molecular-weight immunoglobulin binding protein is now referred to as IbpA and is encoded by the almost 12.3-kb ibpA gene (8). Near the C terminus of IbpA are two direct base pair repeats containing the Fic motif, which has been shown to be cytotoxic for bovine alveolar epithelial cells and phagocytic cells and can cause the cells to round, with disruption of their actin filaments, which may also inhibit phagocytosis (9, 10). Therefore, it could be postulated that H. somni survives within phagocytic cells as a result of compromised cell functions due to cytotoxicity. Although preputial isolates lacking IbpA were highly susceptible to killing by BM cells, a few preputial isolates that produced IbpA (strains 24P, 124P, and 20P) were also killed by BM cells. These results suggest that there may be factors other than IbpA that contribute to the survival of H. somni in phagocytic cells. To determine if IbpA contributed to bacterial survival through host cell toxicity by the Fic domain (9, 19, 20), semipurified IbpA was added to BM cells prior to the addition of strain 129Pt, which lacks IbpA and is highly susceptible to intracellular killing. The addition of IbpA to BM cells enhanced the intracellular survival of strain 129Pt and, to a lesser extent, other H. somni isolates lacking IbpA from the healthy prepuce and did so in a dose-dependent manner. However, the intracellular killing of 129Pt and other strains was not completely abrogated, and significantly higher percentages of strain 129Pt cells were killed by BM cells supplemented with IbpA than strain 2336 cells. Therefore, IbpA may interrupt some essential functions of the phagocytic cells, such as the rearrangement of actin through cytotoxicity (9), but other factors appear to be required to enable H. somni to persist and replicate intracellularly.

The role of IbpA in resistance to killing by serum and phagocytic cells was more comprehensively examined by using Tn and allelic exchange mutants. All Tn mutants tested were capable of replicating in BM cells. However, all the Tn insertions in the ibpA gene were located near the 5′ end of ibpA, which is responsible for immunoglobulin binding through the Fc region. The N terminus of IbpA has homology to the Bordetella pertussis filamentous hemagglutinin (Fha), which contributes to adherence (10, 13), and this region was also proposed to be responsible for adherence to epithelial cells by H. somni (7). These Tn mutants are also deficient in biofilm formation (29), for which the first stage is adherence, which is therefore consistent with the involvement of the N terminus in bacterial adherence. In contrast, the Fic motifs are located within the DR1/DR2 regions located near the C terminus of the protein and were still transcribed in the Tn mutants (data not shown). The Tn insertion may also have caused a frameshift that created a new start codon in the middle of ibpA, which is over 12 kbp in size, or the gene remained in frame, enabling Fic to be transcribed.

The Fic motifs within DR1/DR2 of the IbpA protein are associated with the loss of actin filament function, reduced phagocytosis, and cytotoxicity (7). Therefore, we sought to determine if mutations in specific regions of ibpA would negate intracellular survival. Strains that normally lack IbpA and a mutant lacking the entire ibpA gene are not cytotoxic (9), as they also lack the cytotoxic Fic motifs within DR1/DR2 (10). However, whether cell cytotoxicity is associated with the intracellular survival of H. somni has not been examined. Therefore, mutants with in-frame deletions in specific regions of ibpA were examined for intracellular survival as well as for serum resistance, which is also associated with IbpA (7). All mutants tested with deletions in specific sites throughout ibpA, including DR1 and/or DR2 that include the Fic motifs, or a mutant lacking the entire ibpA gene replicated in BM cells as effectively as parent strain 2336. Therefore, cytotoxicity of phagocytic cells due to the Fic motifs did not explain the ability of H. somni to survive within BM cells.

Bacterial pathogens that can survive within professional phagocytes use one or more mechanisms to avoid intracellular killing, such as (i) inhibition of phagosome-lysosome fusion and acidification, (ii) survival within the phagolysosome, (iii) escape from the phagosome prior to lysosome fusion, and (iv) killing or lysis of the phagocytic cell (or phagosome or lysosome) before or after phagocytosis (30, 31). We examined the colocalization of H. somni with intracellular markers to assess trafficking of H. somni within the phagosome. All H. somni strains and mutants tested that could survive or were killed within BM cells colocalized with EEA-1, which is an early endosomal marker and an early component of phagosome maturation and lysosome fusion (32). However, strain 2336 and other strains that survived within BM cells, whether expressing IbpA or not, failed to acidify phagosomes and did not colocalize with LAMP-2, which is a lysosome-associated membrane protein (32) and an indicator of phagosome-lysosome fusion. The addition of IbpA to BM cells prior to the addition of strain 129Pt did not alter the acidification of the phagosome or colocalization with LAMP-2, further indicating that Fic cytotoxicity was not responsible for the differences in intracellular trafficking noted between strains susceptible and those resistant to intracellular killing. The presence of IbpA and the DR1/DR2 regions has also been associated with inhibition of particle uptake by phagocytic cells (7), most likely through disrupting the function of actin filaments (9). However, in these cases, when H. somni cells expressing either IbpA or semipurified IbpA were added to the phagocytic cells and incubated for a period of time, when bacteria (e.g., S. aureus) or microparticles were then added, cytotoxicity was likely responsible for reducing the subsequent uptake of bacteria or particles. However, there did not appear to be any significant or consistent difference in the uptake of H. somni by healthy BM cells, whether the bacteria expressed IbpA or not. Therefore, in addition to the effects of cytotoxicity by Fic on phagocytic cells, most H. somni strains also appear to be capable of intracellular survival through inhibition of phagosome-lysosome fusion.

H. somni is not unique among mucosal pathogens in being capable of surviving within phagocytic cells. Nontypeable Haemophilus influenzae is capable of surviving within human THP-1 monocytic cells (33), and some strains of Neisseria gonorrhoeae and Neisseria meningitidis are capable of surviving and thriving within polymorphonuclear leukocytes (34). These bacteria have not been classified as facultative intracellular pathogens because their preferred niche within the host is not normally within phagocytic cells. Therefore, the term permissive intracellular pathogen may be a more appropriate term for H. somni and other typically extracellular mucosal pathogens capable of surviving within professional phagocytes. Whether inhibition of the oxidative burst by H. somni bacteria (18) is related to their ability to avoid maturation of the phagolysosome, or is an additional mechanism of intracellular survival, has yet to be determined, as has the role of intracellular survival in the pathogenesis of H. somni diseases.

The expression of IbpA in H. somni isolates is also associated with serum resistance (26, 35), as are the structure of LOS and LOS modification with factors such as sialic acid and phosphorylcholine (36, 37). The association of the entire IbpA protein with serum resistance was confirmed in this study. The isogenic mutant lacking the entire ibpA gene was significantly more serum sensitive than parent strain 2336 at some dilutions of antiserum. However, the lack of only DR1/DR2 did not increase serum sensitivity and in fact made the bacteria more serum resistant. IbpA was originally identified as an immunoglobulin binding protein, and it is likely that binding of host immunoglobulin through the Fc region may inhibit complement binding and activation (35). Why the lack of DR1/DR2 may enhance serum resistance is unknown but could be the result of a conformational change in the protein on the cell surface that further blocks complement binding and activation.

MATERIALS AND METHODS

Bacterial strains.

The H. somni strains and mutants used in this study are listed in Table 1. Mutant 2336ΔIbpA1 has almost the entire ibpA gene replaced with a kanamycin resistance (Kn^r) gene (9), mutant 2336ΔIbpA5 has an in-frame deletion of the 3′-terminal sequence of ibpA (AC region; nucleotides 11725 to 12417 of ibpA), mutant 2336ΔIbpA7 has an in-frame deletion of the ibpA DR2 sequence (nucleotides 10258 to 11439 of ibpA), mutant 2336ΔIbpA8 has an in-frame deletion of the ibpA DR1 sequence (nucleotides 8980 to 10185 of ibpA), mutant 2336ΔIbpA9 has an in-frame deletion of both the DR1 and DR2 sequences (nucleotides 8980 to 11439 of ibpA), and mutant 2336ΔIbpA11 has an in-frame deletion of the ibpA R1R2 sequence (nucleotides 6748 to 8187 of ibpA) (8, 38, 39). Preputial isolate 129Pt does not produce IbpA and is not cytotoxic for epithelial cells (19). However, no other differences have been identified in the outer membrane proteins examined (40). All strains were grown on brain heart infusion (BHI) agar with 5% sheep blood in 5% CO₂ overnight from frozen stocks. The colonies were transferred to BHI broth supplemented with 1% yeast extract, 0.1% Trizma base, and 0.01% TMP (BHIY-TT) (41) and shaken rapidly (∼200 rpm) at 37°C to mid-log phase.

Isolation of BPBMs and cell lines.

Peripheral blood was collected from the jugular vein of Holstein cows into an EDTA-coated tube. Control experiments demonstrated that incubation of H. somni strain 129Pt with each preparation of BPBMs resulted in very similar killing of strain 129Pt (±<5% difference). The buffy coat layer was separated from the red blood cells and plasma by centrifugation at 1,000 × g for 30 min at 15°C, diluted with Hanks' balanced salt solution (HBSS; Life Technologies, Carlsbad, CA), and laid over Ficoll-Paque (Pharmacia, Piscataway, NJ). The BPBMs were recovered following centrifugation, according to the manufacturer's instructions. The viability of the isolated BPBMs was >95%, as determined by trypan blue staining, and the cells were grown as confluent monolayers in RPMI 1640 medium supplemented with 10% fetal bovine serum. Wright stain and careful morphological examination were used to differentiate other white blood cells from BPBMs, which consistently made up about 95% of the cells recovered by Ficoll-Paque. The BPBMs were cultured overnight in 6-well tissue culture plates to allow the cells to adhere to the wells, and the medium was then changed prior to the addition of bacteria or IbpA. The BPBMs were maintained in RPMI 1640 medium with 10% fetal bovine serum and were not stimulated or incubated long enough to differentiate into macrophages.

Primary bovine monocytic cells (BMs) were originally obtained from John Dame, University of Florida (42), and provided to us by David Lindsay, Virginia Tech. The BM cells were derived from the ATCC CRL-6017 cell line (discontinued) and originated from the lymph node of a 2-day-old cow. The cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 2 mM l-glutamine in 5% CO₂ at 37°C (43). The cells were cultured in wells of 6-well plates at 3 × 10⁵ cells/well (in 5 ml) and allowed to adhere for 24 h, and the monolayers were washed 3 times with phosphate-buffered saline (PBS) (pH 7.2) before the addition of IbpA or bacteria. Other cell lines tested in the same manner were mouse J774A.1, human THP, and bovine FBM-117 (44).

Construction of ibpA allelic exchange and transposon mutants.

Seven Tn mutants (mutants 9, 3, 13, 91, 27, 23, and 137) with a Tn insertion within ibpA were selected from a bank of mutants made with the EZ::Tn5⟨KAN-2⟩Tnp transposome (Epicentre, Chicago, IL) as previously described (29). The Tn insertion site was confirmed by sequencing the ends of the Tn and the flanking chromosomal region. Deletion mutant 2336ΔIbpA1 was made by replacement of essentially the entire ibpA gene with a Kn^r gene (9). Allelic exchange mutants 2336ΔIbpA5, 2336ΔIbpA7, 2336ΔIbpA8, 2336ΔIbpA9, and 2336ΔIbpA11 were made by in-frame deletions using a temperature-sensitive plasmid (38, 39).

Preparation of IbpA.

The IbpA protein secreted into the culture medium was concentrated as previously described, with minor modifications (13). Briefly, H. somni strain 2336 was grown in 300 ml of BHIY-TT to mid-log phase. The bacteria were removed by centrifugation at 8,000 × g for 10 min. The supernatant was filtered through a 0.2-μm filter to remove any residual bacteria and then concentrated to 15 ml (20-fold) through a 10,000-molecular-weight (MW) Centriprep centrifugation filter unit (Millipore, Billerica, MA) by centrifugation at 4,000 × g for 30 min at 4°C. The retentate was used as a source of concentrated IbpA in the phagocytosis assay. The purity of IbpA could not be determined because the protein is very large and consists of subunits or aggregates of 76, 120, 270, and 370 kDa (7). More than 20 proteins from 76 kDa to 370 kDa are present in IbpA-positive strains, as determined by Western blotting, but none are present in IbpA-negative strains (19), indicating that this procedure yields predominately IbpA.

Phagocytosis assay.

Bacteria in mid-log phase were coincubated with BPBM or BM cells for 1 h at a multiplicity of infection of 100:1 (bacteria to monocytes). The monocytes were then incubated with 50 μg/ml of gentamicin for 30 min to kill extracellular bacteria, washed three times with PBS, and incubated at 37°C. After incubation for 0 h (1 h after the addition of bacteria and 30 min after the addition of gentamicin), 24 h, 48 h, or 72 h, the monocytes were lysed with distilled water and neutralized with 2× PBS, and the lysate was cultured on BHI blood agar to determine the number of viable intracellular bacteria. The uptake of H. somni by the monocytes was determined at time zero, following lysis of the monocytes and inoculation onto BHI blood agar. The ability of IbpA to protect bacteria from intracellular killing by the monocytes was determined by coincubation of the cells with 4-fold- or 20-fold-concentrated IbpA (3.6 mg/ml) from the culture supernatant for 2 h before coincubation with the bacteria.

Confocal microscopy.

To determine the intracellular trafficking of the bacteria in BM cells, two groups of BM cells were incubated with bacteria for different time periods. The first group of BM cells was incubated with the bacteria for 30 min at 37°C. The culture medium with nonadherent bacteria was removed by washing the monolayer gently three times with PBS, and the medium was replaced with 3% paraformaldehyde for 10 min to fix the cells. Another group of BM cells was also incubated with the bacteria as described above, but after removal of the culture medium, fresh medium was added, and incubation was continued for 3 h at 37°C. The BM cells were then washed and fixed as described above for 10 min. The cells were then permeabilized for 30 min with a 0.1% saponin solution containing 1% bovine serum albumin (BSA) and 5% goat serum. The intracellular H. somni bacteria and phagosomal/lysosomal markers were labeled with rabbit antibodies to H. somni, EEA-1, or LAMP-2 and visualized with goat anti-rabbit antibodies conjugated with Alexa Fluor 488 (H. somni) or Alexa Fluor 546 (markers) (Life Technologies, Carlsbad, CA) by confocal microscopy (Zeiss LSM 510 Meta; Carl Zeiss, Thornwood, NY). The nucleus of the monocytes was visualized with DAPI (4′,6-diamidino-2-phenylindole; Life Technologies, Carlsbad, CA). To determine phagosome acidification, monocytes infected with H. somni were incubated with LysoTracker (Molecular Probes, Eugene, OR) for 1 h, according to the manufacturer's instructions. The cells were then fixed as described above. The percentage of H. somni bacteria that colocalized with the marker was determined as the number of H. somni bacteria that colocalized with either EEA-1 or LAMP-2 divided by the total number of H. somni cells counted. The markers and intracellular H. somni bacteria were identified by SpotFinder Z and counted manually in 5 fields; each field contained at least 4 to 5 monocytes (45, 46). Quantification of the level of each marker, including LysoTracker, was performed by using ImageJ software (https://imagej.nih.gov/ij/download.html) and Oufti software (http://www.oufti.org/), which analyze the percentage of fluorescent dots and fluorescence signals, respectively.

Statistical analyses.

Two-tailed P values were calculated by using the unpaired t test. A P value of <0.05 was considered significant. Statistical analyses were performed by using InStat 3 software (GraphPad Software, Inc., La Jolla, CA).