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1 Histophilus somni Survives in Bovine Macrophages by Interfering with
2 Phagosome-Lysosome Fusion, but Requires IbpA for Optimal Serum
3 Resistance
4 Yu Pan1, Yuichi Tagawa2, Anna Champion1, Indra Sandal1,¶, and Thomas J. Inzana1,3*
5
6 1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of
7 Veterinary Medicine, Virginia Tech, Life Sciences 1, 970 Washington Street, SW, Blacksburg,
8 VA 24061, U.S.A.
9 2Bacterial and Parasitic Disease Research Division, National Institute of Animal Health,
10 National Agriculture and Food Research Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki
11 305-0856, Japan
12 3Virginia Tech Carilion School of Medicine, Roanoke, VA 24016
13
14 ¶Current Address: Memphis VA Medical Center, 1030 Jefferson Avenue, Memphis TN
15 38104 USA
16
17 *Corresponding Author: Email: [email protected]
18
19 Running title: Intracellular survivial of H. somni
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20 ABSTRACT
21 Histophilus somni survives intracellularly in professional phagocytic cells, but the
22 mechanism of intracellular survival is not understood. The Fic motif within the DR1/DR2 IbpA
23 fibrillar network protein of H. somni is cytotoxic to epithelial and phagocytic cells, which may
24 interfere with the bactericidal activity of these cells. To determine the contribution of IbpA and
25 Fic on resistance to host defenses, strains and mutants that lack all of or a small region of ibpA or
26 DR1/DR2 were tested for survival in bovine monocytic cells and for serum susceptibility. A
27 mutant lacking IbpA, but not DR1/DR2, was more susceptible to killing by antiserum than the
28 parent. H. somni strains expressing IbpA replicated in bovine monocytes for at least 72 hours,
29 and were toxic for these cells. Virulent strain 2336 with transposon insertions or deletions within
30 IbpA remained toxic for bovine monocytes. However, strain 2336 mutants lacking all of ibpA or
31 both DR1/DR2 were not toxic to the monocytes, but survived within the monocytes for at least
32 72 hours. Examination of intracellular trafficking of H. somni with monoclonal antibodies to
33 early and late phagosomal markers indicated that early phagosomal marker EEA-1 colocalized
34 with both disease isolate strain 2336 and serum-sensitve mucosal isolate strain 129Pt, but only
35 strain 2336 did not co-localize with late lysosomal marker LAMP-2 and prevented acidification
36 of phagosomes. These results indicate that virulent isolates of H. somni are capable of surviving
37 within phagocytic cells through interference of phagosome-lysosome maturation. Therefore, H.
38 somni may be considered a permissive intracellular pathogen.
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39
40 Key words: Histophilus somni, phagocytosis, IbpA, monocytes, Fic motif, bactericidal,
41 intracellular bacteria
42
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43 The gram-negative bacterium Histophilus somni is an opportunistic pathogen associated with
44 bovine respiratory disease and multi-systemic diseases in cattle and sometimes sheep, including
45 thrombotic meningoencephalitis (TME), myocarditis, arthritis, mastitis, reproductive failure and
46 abortion, and others; probably resulting from bacteremia (1). However, some strains of H. somni
47 are serum-sensitive, and at least one such strain (129Pt) lacks many of the virulence factors
48 associated with disease isolates (2). The only known reservoir for H. somni are the mucosal sites of
49 ruminants (3).
50 Virulent strains of H. somni possess a wide variety of physiological properties and
51 mechanisms that primarily protect the bacteria from host defenses or modulate host immune cells.
52 Such mechanisms include phase variation of lipooligosaccharide (LOS), modification of LOS
53 with sialic acid and phosphorylcholine (4), apoptosis of endothelial cells and neutrophils with
54 disruption of intercellular junctions (5), and biofilm formation (6). Furthermore, the bacteria
55 secrete a fibrillar and surface-associated immunoglobulin binding protein (IbpA), of which the
56 N-terminus region is capable of binding immunoglobulins through their Fc component, and may
57 also mediate adherence of the bacteria to host cells (7).The COOH-terminus of IbpA has
58 homology to a region in YopT in Yersinia spp., but lacks cytotoxic activity (8). In contrast,
59 sequence analysis of ibpA indicates that there are two direct repeats (DR1 and DR2) just upstream
60 of the the yopT-like region, both of which contain a filamentation-induced by c-AMP (Fic) motif:
61 HPFxxGNGR (8). These Fic domains can be found in both bacterial and eukaryotic cells . In H.
62 somni, the Fic motifs of both DR1 and DR2 have been shown to be toxic for bovine epithelial and
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63 phagocytic cells, resulting in rounding up of the cells, increased detachment of infected
64 macrophages, and disruption of actin fibers (9, 10). H. somni strain 2336 can inhibit phagocytosis
65 of microspheres by primary bovine monocytes, but a mutant with essentially the entire ibpA gene
66 deleted can not (9). Antibodies to the recombinant DR2 region of IbpA neutralize the cytotoxic
67 effect on these cells (11). Immunization of mice and calves with recombinant DR2 also protects
68 the animals from H. somni bacteremia and pneumonia, respectively (12, 13). The presence of IbpA
69 on H. somni strains is also associated with serum resistance (7).
70 Virulent strains of H. somni are capable of surviving within bovine polymorphonuclear
71 leukocytes (PMNs), monocytes, and macrophages (14, 15). Phagocytic cells infected with live H.
72 somni are less capable of internalizing a secondary target, such as opsonized Staphylococcus
73 aureus and microspheres (16, 17). Killed, whole bacteria or supernatant from heat-killed bacteria
74 can also inhibit the internalization of S. aureus by PMNs, but not bovine macrophages (16, 17).
75 We have previously reported that the oxidative burst generated by phagocytic cells in contact with
76 viable disease isolates of H. somni is significanlty inhibited. However, there is no inhibition of the
77 oxidative burst by killed H. somni, nonvirulent mucosal strain 129Pt, and heterologous strains that
78 include Haemophilus influenzae and Brucella abortus (18). The mechanism by which H. somni
79 survives within phagocytic cells remains unclear. Because the Fic motifs within IbpA are toxic to
80 phagocytic cells and induce disruption of actin filaments, it is possible that H. somni survives
81 intracellular killing through Fic-mediated interference of phagocytotic cell functions. In this study,
82 we used various mutants with transposon (Tn) insertions and in-frame deletions in ibpA to
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83 examine the contribution of IbpA and the Fic motifs to serum susceptibility and intracellular
84 killing of H. somni, and how virulent disease isolates and avirulent isolates traffic within bovine
85 monocytes.
86
87 RESULTS
88 Intracellular survival of H. somni in bovine monocyte (BM) and bovine peripheral blood
89 monocyte cells (BPBM). Several macrophage or monocyte cell lines, including BM, bovine
90 FBM-17, mouse J774A.1, and human THP cells, were examined for the capability of H. somni
91 strain 2336 to survive intracellularly in comparison to freshly collected BPBM (data not shown).
92 The BM cell line was found to be most comparable to BPBM in regard to intracellular survival or
93 killing of H. somni. H. somni pathogenic strain 2336 survived in BPBM and was cytotoxic to these
94 cells resulting in detachment and rounding up of the cells (data not shown), as previously
95 described for FBM-17 cells (9). In contrast mucosal strain 129Pt from the healthy prepuce was not
96 cytotoxic and did not survive in BM cells (Fig. 1). Strain 2336 was also capable of replicating in
97 the BM cell line, but strain 129Pt was not, which was similar to the results obtained with PMBC
98 (Fig. 1). Mouse macrophage cell line J774.1 and human leukemic cell line THP-1 were also tested,
99 but were unable to kill strain 129Pt (data not shown). Therefore, BM cells were used for all
100 subsequent studies.
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101 Several H. somni strains from disease sites and healthy mucosal sites were tested for their
102 ability to survive within BM cells following phagocytosis. The intracellular number of all isolates
103 from disease sites and some isolates from healthy genital sites increased after 24 h co-incubation
104 with the monocytes (Fig. 2). The Fic domains within DR1/DR2 of IbpA have been shown to be
105 cytotoxic to host cells (9), and the presence or absence of ibpA in all the H. somni strains used in
106 this study has been previously documented (Table 1) (19). All the disease isolates and most of the
107 vaginal isolatess tested were able to replicate intracellularly, but most preputial isolates tested did
108 not (Table 1 and Fig. 2). Strains 1P, 129Pt, 130Pf, and 133P from the bovne prepuce do not
109 produce IbpA (19) and were unable to replicate intracellularly after 24 h co-incubation (Table 1
110 and Fig. 2). However, some preputial isolates previously shown to produce IbpA (24P, 124P, and
111 20P) were also killed by BM cells, though preputial IbpA-producing strain 22P was resistant. Of
112 interest was that strain 1225, which was isolated from the bovine prepuce in The Netherlands, was
113 highly resistant to intracellular killing, but it was unknown whether this isolate was associated with
114 disease or expressed IbpA. Therefore, the expresion of IbpA was not universally associated with
115 intracellular survival.
116 The role of IbpA in intracellular survival of H. somni. To assess the direct effect of IbpA
117 on H. somni intracellular survival, BM cells were incubated for 2 hrs with H. somni culture
118 supernatant concentrated 1:4 or 1:20 to enrich for IbpA, then infected with H. somni strain 129Pt,
119 which cannot survive intracellularly. After 24 h incubation the intracellular survival of strain
120 129Pt in BM cells incubated with culture supernatant concentrated 1:4 or 1:20 was significantly (p
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121 < 0.001) greater than for the bacteria within monocytes not incubated with IbpA, and this effect
122 was dose-dependent (Fig. 3). These results indicated that IbpA had a negative effect on BM cells to
123 subsequently take up and/or kill strain 129Pt. IbpA (20-fold concentrate) also significantly
124 enhanced the intracellular survival of the other H. somni strains (24 h incubation in monocytes)
125 that were not as susceptible to intracellular killing as strain 129Pt, but also lacked the ibpA gene
126 (1P, 133P, and 130Pf; p = 0.004, 0.001, and 0.005, respectively), although not to the extent as for
127 strain 129Pt (Fig. 4). However, the addition of IbpA only moderately enhanced intracellular
128 survival of strain 24P and, had no effect on strains 124P and 20P, (Fig. 4), all of which, as expected,
129 produce IbpA (19).
130 Survival of H. somni ibpA mutants in BM cells. Several mutants with Tn insertion
131 mutations in ibpA were selected from a bank of Tn mutants for intracellular survivial in BM cells.
132 All the mutants replicated significantly more slowly than the parent strain at 24 h post-incubation
133 with BM cells (p < 0.001), but none of the mutants demonstrated a significant difference in the
134 number of viable intracellular bacteria after 48 or 72 h of incubation (p > 0.05) (data not shown).
135 However, all of these mutants contained the Tn in the region encoding for Fc binding by IbpA, and
136 none contained the tranposon within the DR1/DR2 region. Mutants with the ibpA gene replaced
137 with a KnR gene or with in-frame deletions in ibpA were also tested for intracellular survival (Fig.
138 5). Mutants with essentially the entire ibpA gene removed (2336ΔIbpA1) or both DR1 and DR2
139 (DR1/DR2) containing the Fic motifs (2336ΔIbpA9) were capable of surviving within BM cells as
140 effectively as parent strain 2336. Strains 2336ΔIbpA5 (deletion near 3’-terminus), 2336ΔA7 (DR2
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141 only deleted), 2336ΔA8 (DR1 only deleted), and 2336ΔA11 (deletion of R1R2 sequences) were
142 also no more susceptible to intracellular killing than the parent (data not shown). Therefore,
143 destruction of actin filaments and cell toxicity due to the Fic motifs were not, by themselves, the
144 mechanism by which H. somni survived within phagocytic cells. Furthermore, there was not a
145 significant difference between the presence or absence of IbpA, DR1/DR2, and uptake of the
146 bacteria.
147 Intracellular trafficking of H. somni within BM cells. Intracellular trafficking of pneumonia
148 isolate strain 2336 in comparison to mucosal isolate strain 129Pt was examined by confocal
149 microscopy to further clarify the mechanism of H. somni intracellular survival. Early phagosomal
150 marker EEA-1 and late lysosomal marker LAMP-2 were both expressed in BM and co-localized
151 with strain 129Pt with and without the addition of IbpA, and with strain 2336. Phagolysosomes
152 containing strain 129Pt with and without the addition of IbpA were also acidified and co-localized
153 with LAMP-2 (Figs. 6 and 7, p > 0.05). Therefore, intracellular trafficking of strain 129Pt in BM
154 was not affected by the addition of IbpA. However, although EEA-1 co-locallized with strain 2336,
155 the acidification of phagolysosomes and the expression/co-localization of LAMP-2 in BM cells
156 infected with strain 2336 was significantly lower than with strain 129Pt (Figs. 6 and 7, p = 0.008).
157 The vaginal isolates used in this study were also capable of surviving in BM, although these
158 isolates were not associated with disease. To compare the intracellular trafficking of a typical
159 vaginal isolate to disease isolate strain 2336, strain 64Vc was also examined by confocal
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160 microscopy. Strain 64Vc co-localized with early phagosomal marker EEA-1 10 min after infection,
161 but also inhibited the expression of LAMP-2 (p = 0.01) and the acidification of the phagosomes (p
162 = 0.001 compared to 129Pt) (Figs. 6 and 7).
163 Serum susceptibility of IbpA and DR1/DR2 mutants. Histophilus somni expression of IbpA
164 is also asociated with serum resistance (7). Therefore, we sought to determine if a normally serum
165 resistant strain becomes serum sensitive with the loss of IbpA or the DR1/DR2 regions. Serum
166 resistance is relative in H. somni; even “serum resistant” strains can be killed in the presence of
167 adequate antibodies to H. somni surface antigens. Therefore, to maximize the serum bactericidal
168 effect, antiserum to H. somni LOS was used in the presence of pre-colostral calf serum as an
169 antibody-free source of complement. In the absence of any antiserum, strain 2336 and mutants
170 lacking essentially all of IbpA (H. somniΔIbpA1) or both DR1 and DR2 (H. somniΔIbpA9)
171 increased in numbers, indicating these bacteria were resistant to the effects of complement alone
172 (Fig. 8). At 40% antiserum, all the strains were effectively killed. However, in the presence of
173 between 10% and 30% antiserum mutant H. somniΔ2336IbpA9, lacking DR1/DR2, was
174 significantly more resistant to killing than even strain 2336 (p <0.008), but H. somni mutant
175 2336ΔIbpA1 (lacking all of IbpA) was more susceptible to killing in 10% antiserum (p = 0.004)
176 than strain 2336, though not as susceptible as strain 129Pt. Therefore, the IbpA protein, but not the
177 DR1/DR2 repeats containing the Fic motifs, contributed to serum resistance. However, other
178 factors that may also be deficient in strain 129Pt appear to contribute to serum resistance in H.
179 somni.
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180
181 DISCUSSION
182 Virulent strains of H. somni are readily phagocytosed, but not killed, by neutrophils,
183 macrophages, or monocytic cells (14, 15, 20). However, the mechanism by which H. somni surives
184 within these cells is not clear. Generation of reactive oxygen intermediates are an important
185 defense mechanism that phagocytic cells use to kill bacteria following phagocytosis (21).
186 Inhibition of the oxidative burst by phagocytic cells following incubation with H. somni has been
187 well established and may contribute to intracellular survival (14, 17, 18, 22-24). Several
188 investigators have shown that this inhibition of the oxidative burst requires contact with, or the
189 presence of, viable H. somni (16, 18, 22, 24), whereas others have reported such inhibitory activity
190 can occur by killed cells or cell fractions (17, 25). The reason for this difference is unclear, but may
191 be related to differences in the assays used. Furthermore, while disease isolates of H. somni are
192 very efficient at inhibiting the oxidative burst of phagocytic cells, serum-sensitive isolates from
193 the normal bovine prepuce are not (18). In addition, incubation of phagocytic cells with H. somni
194 inhibits their subsequent uptake of opsonized S. aureus (16, 17, 22), indicating that the cells have
195 been compromised in regard to phagocytic capacity following incubation with H. somni.
196 Bovine BM cells killed strain 129Pt as effectively as BPBM, but bovine FBM-17 cells, murine
197 J774A.1, and human THP cells did not, indicating that the BM cells are well adapted to H. somni
198 or that some immortalized cell lines may be deficient in aspects of intracellular killing. Strain 2336
199 survived within BM cells as well as in BPBM, indicating this cell line was a suitable surogate for
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200 BPBM for use in these assays. In this study, additional disease and mucosal isolates were tested for
201 intracellular survival. However, while most preputial isolates were confirmed to be less capable of
202 replicating in monocytes, isolates from the healthy vagina were as capable of surviving
203 intracellularly as disease isolates. Of interest was that the isolates resistant to intracellular killing
204 are also serum-resistant (19, 26).
205 Extracellular protein toxins have not been identified in H. somni. However, high molecular
206 weight fibrillar proteins that bind IgG2 are present on the cell surface of all H. somni strains tested
207 except for some preputial isolates (19, 27, 28). This high molecular size, fribillar immunoglubulin
208 binding protein is now referred to as IbpA, and is encoded by the almost 12.3 kb ibpA gene (8).
209 Near the C-terminus of IbpA are two direct base pair repeats containing the motif Fic, which has
210 been shown to be cytotoxic for bovine alveolar epithelial cells and phagocytic cells, and can cause
211 the cells to round up and their actin filaments disrupted, which may also inhibit phagocytosis (9).
212 Therefore, H. somni may be able to survive within phagocytic cells as a result of compromised cell
213 functions due to cytotoxic effects. Although the isolates lacking IbpA were highly susceptible to
214 killing by BM cells, a few preputial isolates that did produce IbpA (strains 24P, 124P, and 20P)
215 were also killed by BM cells, suggesting there may be factors other than IbpA that contribute to the
216 resistance of H. somni to killing by phagocytic cells. To determine if IbpA contributed to bacterial
217 survival through host cell toxicity by the Fic domain (9, 19, 20), semi-purified IbpA was added to
218 the BM cells prior to addition of strain 129Pt, which lacks IbpA and is highly susceptible to
219 intracellular killing. The addition of IbpA to BM cells did enhance the intracellular survival of
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220 strain 129Pt, and to a lesser extent other H. somni isolates lacking IbpA from the healthy prepuce,
221 and did so in a dose-dependent manner. However, the intracellular killing of 129Pt and other
222 strains was not completely abrogated, and significantly more cells of strain 129Pt were killed by
223 BM cells supplemented with IbpA than strain 2336. Therefore, IbpA may interrupt some essential
224 functions of the phagocytic cells, such as the rearrangement of actin through cytotoxicity (9), but
225 other factors appear to be required to enable H. somni to replicate intracellularly.
226 To more comprehensively examine the role of IbpA in resistance to killing by serum and
227 phagocytic cells, Tn and allelic exchange mutants were tested for intracellular survival. All ibpA
228 and other Tn mutants tested were capable of replicating in BM cells. However, all the Tn insertions
229 in the ibpA mutants were located near the 5’ end of the ibpA gene, which is responsible for
230 immunoglubulin binding through the Fc region. The N-terminus of IbpA has homology to the
231 Bordetella pertussis filamentous hemagglutinin (Fha), which contributes to adherence (10, 13),
232 and this region is also proposed to be responsible for epithelial cell adherence by H. somni (7).
233 These Tn mutants are also deficient in biofilm formation (29), for which the first stage is adherence,
234 and is therefore consisten with a role of the N-terminus in bacterial adherence. In contrast, the Fic
235 motifs are located within the DR1/DR2 repeats located near the C-terminus of the protein and were
236 still transcribed in the Tn mutants (data not shown). The Tn insertion may also have caused a frame
237 shift that created a new start codon in the middle of ibpA, which is over 12 kbp in size, or the gene
238 remained in frame enabling Fic to be transcribed.
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239 The Fic motifs within DR1/DR2 of the IbpA protein are associated with loss of actin filament
240 function, reduced phagocytosis, and cytotoxicity (7). Therefore, we sought to determine if
241 mutations in specific regions of ibpA would negate intracellular survival. Strains that normally
242 lack IbpA or a mutant lacking all of the ibpA gene are not cytotoxic (9), as they also lack the
243 cytotoxic Fic motifs within DR1/DR2 (10). However, whether cell cytotoxicity is associated with
244 intracellular survival of H. somni has not been examined. Therefore, mutants with in-frame
245 deletions in specific regions of ibpA were examined for intracellular survival as well as serum
246 resistance, which is also associated with IbpA (7). All mutants tested with deletions in specific
247 sites throughout ibpA, including DR1 and/or DR2 that include the Fic motifs, or a mutant lacking
248 the entire ibpA gene replicated in BM cells as effectively as parent strain 2336. Therefore,
249 cytotoxicity of phagocytic cells due to the Fic motifs do not explain the capability of H. somni to
250 survive within BM cells.
251 Bacterial pathogens that can survive within professional phagocytes use one or more
252 mechanisms to avoid intracellular killing, such as: 1) inhibition of phagosome-lysosome fusion
253 and acidification; 2) survival within the phago-lysosome; 3) escape from the phagosome prior to
254 lysosome fusion; 4) killing or lysing of the phagocytic cell (or phagosome or lysosome) before or
255 after phagocytosis (30, 31). We examined co-localization of H. somni with intracellular markers to
256 asses traficking of H. somni in the phagosome. All H. somni strains and mutants tested that could
257 survive or were killed within BM cells co-localized with EEA-1, which is an early endosomal
258 marker, and an early component in phagosome maturation and lysosome fusion (32). However,
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259 strain 2336 and other strains that survived within BM cells, whether expressing IbpA or not, failed
260 to acidify phagosomes and did not co-localize with LAMP-2, which is a lysosome-associated
261 membrane protein (32), and an indicator of phagosome-lysosome fusion. The addition of IbpA to
262 BM cells prior to addition of strain 129Pt did not alter acidification of the phagosome or
263 co-localization with LAMP-2, further indicating that Fic toxicity was not responsible for the
264 differences in intracellular traficking noted between strains susceptible or resistant to intracellular
265 killing. The presence of IbpA and DR1/DR2 regions has also been associated with inhibition of
266 uptake by phagocytic cells (7) most likely through disrupting the function of actin filaments (9).
267 However, in these cases either H. somni cells expressing IbpA or semi-purified IbpA were added
268 to the phagocytes, incubated for a period of time, and then bacteria (e.g. S. aureus) or
269 microparticles added. In these cases, cytotoxicity was likely responsible for reducing subsequent
270 uptake of bacteria or particles. However, there did not appear to be any significant or consistent
271 difference in the uptake of H. somni by healthy BM cells, whether the bacteria expressed IbpA or
272 not. Therefore, in addition to the effects of cytotoxicity by Fic on phagocytic cells, most H. somni
273 strains also appear to be capable of intracellular survival through inhibition of
274 phagosome-lysosome fusion.
275 H. somni is not unique among mucosal pathogens in being capable of surviving within
276 phagocytic cells. Nontypable Haemophislus influenzae is capable of surviving within human
277 THP1 monocytic cells (33), and some strains of Neisseria gonorrhoeae and N. meningitidis are
278 capable of surviving and thriving within polymorphonuclear leukocytes (34). These bacteria have
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279 not been classified as facultative intracellular pathogens because their preferred niiche within the
280 host is not normally within phagocytic cells. Therefore, the term permissive intracellular pathogen
281 may be a more appropriate term for H. somni and other typically extracellular mucosal pathogens
282 capable of surviving within professional phagocytes. Whether inhibition of the oxidative burst by
283 H. somni (18) is related to their capability to avoid maturation of the phago-lysosome or is an
284 additional mechanism of intracellular survival has yet to be determined, as does the role of
285 intracellular survival in the pathogenesis of H. somni diseases.
286 The expression of IbpA on H. somni isolates is also associated with serum resistance (26, 35),
287 as is the structure of the LOS oligosaccharide, and its modification with factors such as sialic acid
288 and phosphorylcholine (36, 37). The association of the entire IbpA protein with serum resistance
289 was confirmed in this study. The isogenic mutant lacking the entire ibpA gene was significnatly
290 more serum-sensitive than parent strain 2336 at some dilutions of antiserum. However, the lack of
291 only DR1/DR2 did not increase serum sensivitiy,but in fact made the bacteria more
292 serum-resistant. IbpA was originally identified as an immunoglubulin binding protein, and it is
293 likely that binding of host immunoglobulin through the Fc region may inhibit complement binding
294 and activation (35). Why the lack of DR1/DR2 may enhance serum reistance is unknown, but
295 could be the result of a conformational change in the protein on the cell surface that further blocks
296 complement binding and activation.
297
298 MATERIALS AND METHODS
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299 Bacterial strains. The H. somni strains and mutants used in this study are listed in Table 1.
300 Mutant 2336ΔIbpA1 has almost all of ibpA replaced with a kanamycin resistance gene (9), mutant
301 2336ΔIbpA5 has an in-frame deletion of the 3'-terminal sequence of ibpA (AC region; nucleotides
302 11725-12417 of ibpA), mutant 2336ΔIbpA7 has an in-frame deletion of the ibpA DR2 sequence
303 (nucleotides 10258-11439 of ibpA), mutant 2336ΔIbpA8 has an in-frame deletion of the ibpA DR1
304 sequence (nucleotides 8980-10185 of ibpA), mutant 2336ΔIbpA9 has an in-frame deletion of both
305 the DR1 and DR2 sequences (nucleotides 8980-11439 of ibpA), and mutant 2336ΔIbpA11 has an
306 in-frame deletion of the ibpA R1R2 sequence (nucleotides 6748-8187 of ibpA) (8, 38, 39).
307 Preputial isolate 129Pt does not produce IbpA and is not cytotoxic for epithelial cells (19).
308 However, no other differences have been identified in outer membrane proteins examined (40). All
309 strains were grown on Brain Heart Infusion (BHI) agar with 5% sheep blood in 5% CO2 overnight
310 from frozen stocks. The colonies were transferred to BHI broth supplemented with 1% yeast
311 extract, 0.1% Trizma base, and 0.01% thiamine monophosphate (TMP) (BHIY-TT) (41), and
312 shaken rapidly (~200 rpm) at 37 °C to mid-log phase.
313 Isolation of BPBM and cell lines. Peripheral blood was collected from the jugular vein of
314 Holstein cows into an EDTA-coated tube. Control experiments demonstrated that incubation of H.
315 somni strain 129Pt with each preparation of BPBM resulted in very similar killing of strain 129Pt
316 (+<5% difference). The buffy coat layer was separated from the red blood cells and plasma by
317 centrifugation at 1000 x g for 30 min at 15 °C, diluted with Hank’s Balanced Salt Solution (HBSS;
318 Life Technologies, Carlsbad, CA) and laid over Ficoll-Paque (Pharmacia, Piscataway, NJ). The
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319 BPBM were recovered following centrifugation, as per the manufacturer’s instructions. The
320 viability of the isolated BPBM was greater than 95%, as determined by trypan blue staining, and
321 were grown as confluent monolayers in RPMI-1640 medium supplemented with 10% fetal bovine
322 serum. A bovine monocytic cell line (BM) derived from blood monocytes of a 6-year-old
323 Guernsey cow was originally obtained from Dr. John Dame, University of Florida, and provided
324 by Dr. David Lindsay, Virginia Tech. The cells were grown in RPMI-1640 medium supplemented
325 with 10% heat-inactivated fetal bovine serum and 2 mM L-glutamine in 5% CO2 at 37 °C (42). The
326 cells were cultured in wells of 6-well plates at 3x105/well (in 5 ml), allowed to adhere for 24 h, and
327 the monolayers were washed 3 times with phosphate buffered saline, pH 7.2 (PBS) before adding
328 IbpA or bacteria. Other cell lines tested in the same manner were mouse J774A.1, human THP, and
329 bovine FBM-117 (43).
330 Construction of ibpA allelic exchange and transposon (Tn) mutants. Seven mutants (#9,
331 #3, #13, #91, #27, #23 and #137) with a Tn insertion within ibpA were selected from a bank of
332 mutants made with EZ::Tn5™
333 previously described (29). The Tn insertion site was confirmed by sequencing the ends of the Tn
334 and the flanking chromosomal region. Deletion mutant 2336ΔIbpA1 was made by replacement of
335 essentially all of the ibpA with a kanamycin resistance gene (KnR) (9). Mutants 2336ΔIbpA5,
336 2336ΔIbpA7, 2336ΔIbpA8, 2336ΔIbpA9, and 2336ΔIbpA11 were made by in-frame deletions
337 using a temperature-sensitivite plasmid (38, 39).
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338 Preparation of IbpA. The IbpA protein secreted into the culture medium was concentrated as
339 previously described with minor modifications (13). Briefly, H. somni strain 2336 was grown in
340 300 ml of BHIY-TT to mid-log phase. The bacteria were removed by centrifugation at
341 8000 × g for 10 min. The supernatant was filtered through a 0.2-μM filter to remove any residual
342 bacteria and then concentrated to 15 ml (20-fold) through a 10,000 MW centriprep centrifugation
343 filter unit (Millipore, Billerica, MA) by centrifugation at 4,000 × g for 30 min at 4°C. The
344 retentate was used as a source of concentrated IbpA in the phagocytosis assay. The purity of
345 IbpA could not be determined because the protein is very large and consists of subunits or
346 aggregates of 76, 120, 270, and 370 kDa (7). In Western blots more than 20 proteins from 76
347 kDa to 370 kDa are present in IbpA-positive strains, but none are present in IbpA-negative
348 strains, indicating this procedure results in a sample consisting of predominately IbpA (19).
349 Phagocytosis assay. Bacteria in mid-log phase were co-incubated with BPBM or BM
350 monocytic cells for 1 h at a 100:1 multiplicity of infection (bacteria:monocytes). The monocytes
351 were then incubated with 50 μg/ml of gentamicin for 30 min to kill extracellular bacteria, washed
352 three times with PBS, and incubated at 37 °C. After incubation for 0 h (1 h after addition of
353 bacteria and 30 min after addition of gentamicin), 24 h, 48 h, or 72 h the monocytes were lysed
354 with distilled water, neutralized with 2X PBS, and the lysate cultured onto BHI blood agar to
355 determine the number of viable intracellular bacteria. The uptake of H. somni by the monocytes
356 was determined at time 0, following lysis of the monocytes and inoculation to BHI blood agar. The
357 capability of IbpA to protect bacteria from intracellular killing by the monocytes was determined
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358 by co-incubation of the cells with 20-fold concentrated IbpA (3.6 mg/ml), or a sample diluted to
359 represent a 4-fold concentration, from culture supernatant for 2 h before co-incubation with the
360 bacteria.
361 Confocal Microscopy. To determine the intracellular trafficking of the bacteria in BM cells,
362 two groups of BM cells were incubated with bacteria for different time periods. In group 1 BM
363 cells were incubated with the bacteria for 30 min at 37°C, the culture medium with nonadherent
364 bacteria was removed by washing the monolayer gently three times with PBS, and the medium
365 was replaced with 3% paraformaldehyde for 10 min to fix the cells. Another group of BM cells
366 was also incubated with the bacteria as above, but after removal of the culture medium, fresh
367 medium was added and incubation was continued for 3 hr at 37°C. The BM cells were then washed
368 and fixed as above for 10 min. The cells were then permeabilized with 0.1% saponin solution
369 containing 1% bovine serum albumin (BSA) and 5% goat serum for 30 min. The intracellular H.
370 somni and phgosomal/lysosomal markers were labeled with rabbit antibodies to H. somni, or
371 EEA-1 or LAMP-2 and visualized with goat anti-rabbit antibodies conjugated with Alexa Fluor®
372 488 (H. somni) or Alexa Fluor® 546 (markers) (Life Technologies, Carlsbad, CA) by confocal
373 microscopy (Zeiss LSM 510 META; Carl Zeiss, Thornwood N.Y.). The nucleus of the monocytes
374 was visualized with DAPI (Life Technologies, Carlsbad, CA). To determine phagosome
375 acidification, the monocytes infected with H. somni were incubated with Lysotracker (Molecular
376 Probes, Eugene, OR) for 1h, following the manufacturer’s instructions. The cells were then fixed
377 as described above. To determine the percentage of H. somni co-localized with the markers, the
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378 percent of H. somni co-localized with each marker was determined from the number of H.
379 somni co-localized with either EEA-1 or LAMP-2 divided by the total number of H. somni cells.
380 The markers and intracellular H. somni were identified by SpotFinder Z and counted manually in 5
381 fields, each containing at least 4-5 monocytes (44, 45). Quantification of each marker, including
382 LysoTracker, was determined using the software imageJ (https://imagej.nih.gov/ij/download.html)
383 and MicrobeTracker (http://www.microbetracker.org/), which analyze the percentage of
384 fluorescent dots and fluorescence signals, respectively.
385 Statistical analyses. Two-tailed P values were calculated using the unpaired t test. A P value
386 <0.05 was considered significant. Statistical analyses were determined using InStat 3 software
387 (GraphPad Software, Inc., La Jolla, CA).
388
389 ACKNOWLEDGEMENTS
390 We would like to tank Dr. David Lindsay for providing the BM macrophage cell line, Dr.
391 Lynette Corbeil and Vivian Fussing for providing bacterial strains, and Poorna Goswami, Angelea
392 Sadaat, and Gillian Rodgers for excellent technical assistance. This work was supported by
393 USDA-NIFA grant 2013-67015-21314 to TJI and by HATCH funds from the Virginia-Maryland
394 College of Veterinary Medicine.
21 bioRxiv preprint doi: https://doi.org/10.1101/322768; this version posted May 15, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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526
527
528
529
530 Figure legends
531 Figure 1. Survival of H. somni strains 2336 and 129Pt in BPBM and BM monocyte cell line. The
532 bacteria were incubated with monocyte cells for 1 hr (uptake time) at 100:1 (bacteria:monocytes),
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533 gentamicin added to kill extracellular bacteria, and after 30 min the moncytes were washed,
534 incubated for 0, 24, 48, or 72 hr, lysed, and the released bacteria cultured on blood agar. □, strain
535 2336 incubated with BPBM; Δ, strain 129Pt incubated with BPBM; ◊, strain 2336 incubated with
536 BM cells; ○, strain 129Pt incubated with BM cells. Results represent the mean + standard
537 deviation of at least 3 experiments. Standard deviation bars are difficult to see due to their small
538 size and the symbols.
539 Figure 2. Survival of H. somni strains in BM monocytes after 24 h co-incubation. The strains on
540 the left of 100% were killed in the monocytes; the strains on the right of 100% replicated in the BM
541 cells. Percent survival of H. somni was determined by lysis of BM cells after 1 h (incubation time
542 given for uptake) and 24 h post co-incubation, and culture. The number of colonies recovered after
543 24 h incubation was divided by the number of colonies after 1 h of incubation x 100. Results
544 represent the mean + standard deviation of 3 experiments.
545 Figure 3. Percent intracellular survival of H. somni 129Pt after 24 h incubation in BM cells
546 preincubated for 2 hr with or without 20-fold or 4-fold concentrated, semi-purified IbpA from
547 2336 supernatant. Strain 2336 survival shown for comparison. Results represent the mean +
548 standard deviation of at least 3 experiments.
549 Figure 4. Percent survival of H.somni preputial isolates after 24 h incubation in BM cells that have
550 been preincubated for 2 h with 20-fold concentrated culture supernatant containing IbpA. Strains
551 129Pt, 1P, 133P, and 130Pf lack IbpA and were significantly more resistant to intracellular killing
29 bioRxiv preprint doi: https://doi.org/10.1101/322768; this version posted May 15, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
552 by BM monocytes after addition of IbpA. Strains 24P, and 124P and 20P produce IbpA and there
553 was little or no difference, respectively, in killing by BM cells following preincubation of the cells
554 with IbpA. Results represent the mean + standard deviation of at least 3 experiments.
555 Figure 5. Intracellular survival of H. somni ibpA mutants and control strains in BM cells over 72
556 hr. Survival of strain 129Pt (●); strain 2336 (■); strain 2336ΔIbpA1 (▲); strain 2336ΔIbpA9 (♦).
557 The lack of DR1/DR2 repeats containing the toxic Fic motifs or essentially the entire IbpA protein
558 had no effect on itracellular survival of strain 2336 in BM cells. Results represent the mean +
559 standard deviation of at least 3 experiments.
560 Figure 6. Confocal microscopy of H. somni strains 2336, strain 129Pt with and without addition of
561 IbpA, and 64Vc following phagocytosis. The markers EEA-1 (10 min), and LAMP-2 and
562 Lysotracker (3 hr) were stained with Alexa Fluor® 546 (red) and H. somni with Alexa Fluor® 488
563 (green). The red arrowhead point to the markers, the green arrowheads point to H. somni, the
564 yellow arrowheads point to colocalization of the markers with H. somni. Each photo shown is a
565 representative field of 6-10 fields examined from 3 separate experiments.
566 Figure 7. Quantification of H. somni serum resistant strains 2336 and 64Vc, and serum sensitive
567 strain 129Pt (with and without added IbpA) that co-localized with markers EEA-1 (white column),
568 LysoTracker (black column) or LAMP-2 (gray column). The co-localization of H. somni with each
569 marker was quantified at different time points: EEA-1 (10 min), LysoTracker/acidification and
570 LAMP-2 (3 h). Quantification of EEA-1 and LAMP-2 markers, and LysoTracker, was determined
30 bioRxiv preprint doi: https://doi.org/10.1101/322768; this version posted May 15, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
571 using the software imageJ and MicrobeTracker, which analyze the percentage of fluorescent dots
572 and fluorescence signals, respectively. Results represent the mean + standard deviation of at least 3
573 experiments.
574 Figure 8. Serum resistance or susceptibiilty of H. somni and ibpA mutants. Isogenic mutants of
575 control strain 2336 (●; serum resistant) lacking all of ibpA (▲; 2336ΔIbpA1) or only DR1/DR2
576 repeats containing the toxic Fic motifs (▼; 2336ΔIbpA9) were tested for susceptibility to killing
577 by antiserum to H. somni lipooligosaccharide and bovine complement in comparison to control
578 strain 129Pt (■; serum sensitive). Results represent the mean + standard deviation of at least 3
579 experiments.
31 bioRxiv preprint doi: https://doi.org/10.1101/322768; this version posted May 15, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
580 Table 1: H. somni trains used in this study.
581 Strain Description IbpA presenta Serum resistant Reference or source
582 2336b Isolated from bovine pneumonia strain + + (26)
583 2336::TnfhaB #3 ibpA Tn mutant + NDc (29)
584 2336::TnfhaB #13 ibpA Tn mutant + ND (29)
585 2336::TnfhaB #91 ibpA Tn mutant + ND (29)
586 2336::TnfhaB #9 ibpA Tn mutant + NDb (29)
587 2336::TnfhaB #27 ibpA Tn mutant + ND (29)
588 2336::TnfhaB #23 ibpA Tn mutant + ND (29)
589 2336::TnfhaB #137 ibpA Tn mutant + ND (29)
590 2336ΔibpA1 ibpA deletion mutant (all of ibpA) - (9)
591 2336ΔibpA5 ibpA deletion mutant (3'-terminal sequence) ND (38, 39)
592 2336ΔibpA7 ibpA deletion mutant (DR2 sequence) ND (38, 39)
32 bioRxiv preprint doi: https://doi.org/10.1101/322768; this version posted May 15, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
593 2336ΔibpA8 ibpA deletion mutant (DR1 sequence) ND (38, 39)
594 2336ΔibpA9 ibpA deletion mutant (DR1 andDR2 sequence;
595 all of Fic motif) + (38, 39)
596 2336ΔibpA11 ibpA deletion mutant (R1R2 sequence) ND (38, 39)
597 129Pt normal prepuce - - (26)
598 1P normal prepuce - - (26)
599 130Pf normal prepuce - - (26)
600 133P normal prepuce - - (26)
601 124P normal prepuce + - (26)
602 20P normal prepuce + + (26)
603 24P normal prepuce + - (26)
604 221V normal vagina + - (26)
605 22P normal prepuce + + (26)
33 bioRxiv preprint doi: https://doi.org/10.1101/322768; this version posted May 15, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
606 1225 prepuce ND ND V. Fussing, Natl. Vet.
607 Laboratry, Denmark
608 80 normal vagina + + (26)
609 1297 pneumonia + + (26)
610 5166 pneumonia ND ND (26)
611 738 phase variant of 2336
612 isolated from challenged calf lung + + (46)
613 649 abortion + + (26)
614 29Vb normal vagina + - (26)
615 318 phase variat of strain 738 + + (26)
616 8025 TME + + (26)
617 2089 abortion + + (26)
618 41Vc normal vagina + - (26)
34 bioRxiv preprint doi: https://doi.org/10.1101/322768; this version posted May 15, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
619 208V normal vagina + - (26)
620 202V normal vaginal + - (26)
621 570 abortion + - (26)
622 64Vc normal vagina + + (26)
623 aThe presence or absence of ibpA was determined by PCR and the production of IbpA by Western Blotting of the bacteria culture
624 supernatant (19).
625 bAll mutants were derived from strain 2336.
626 cND – not determined.
35 bioRxiv preprint doi: https://doi.org/10.1101/322768; this version posted May 15, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
627
628
629 Fig. 1.
630
36 bioRxiv preprint doi: https://doi.org/10.1101/322768; this version posted May 15, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
631
632 Fig. 2.
633
634
635
636
37 bioRxiv preprint doi: https://doi.org/10.1101/322768; this version posted May 15, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
637
638 Fig. 3.
639
640
641
38 bioRxiv preprint doi: https://doi.org/10.1101/322768; this version posted May 15, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
642
643 Fig. 4.
39 bioRxiv preprint doi: https://doi.org/10.1101/322768; this version posted May 15, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
644
645 Fig. 5.
40 bioRxiv preprint doi: https://doi.org/10.1101/322768; this version posted May 15, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
646
647 Fig. 6.
41 bioRxiv preprint doi: https://doi.org/10.1101/322768; this version posted May 15, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
648
649 Fig. 7.
42 bioRxiv preprint doi: https://doi.org/10.1101/322768; this version posted May 15, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
650
651 Fig. 8.
43