The Coxiella Burnetii Qph1 Plasmid Is a Virulence Factor for Colonizing
bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1 The Coxiella burnetii QpH1 plasmid is a virulence factor for
2 colonizing bone marrow-derived murine macrophages
3
4 Shengdong Luo1,2¶, Zhihui Sun2¶, Huahao Fan1¶, Shanshan Lu1,3&, Yan Hu3&,
5 Ruisheng Li3&, Xiaoping An1&, Yigang Tong1*, Lihua Song1,2*
6
7 1 Beijing Advanced Innovation Center for Soft Matter Science and Engineering, 8 College of Life Science and Technology, Beijing University of Chemical Technology, 9 Beijing, China 10 11 2 State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology 12 and Epidemiology, Beijing, China 13 14 3 Research Center for Clinical Medicine, The Fifth Medical Center of PLA General 15 Hospital, Beijing, China 16
17 * Corresponding author
18 Email: [email protected] (LS); [email protected] (YT).
19
20 ¶ These authors contributed equally to this work.
21 & These authors also contributed equally to this work.
22 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
23 Abstract
24 Coxiella burnetii carries a large conserved plasmid or plasmid-like chromosomally
25 integrated sequence of unknown function. Here we report the curing of QpH1 plasmid
26 from C. burnetii Nine Mile phase II, the characterization of QpH1-deficient C.
27 burnetii in in vitro and in vivo infection models, and the characterization of plasmid
28 biology. A shuttle vector pQGK, which is composed of the CBUA0036-0039a region
29 (predicted for QpH1 maintenance), an E. coli plasmid ori, eGFP and kanamycin
30 resistance genes was constructed. The pQGK vector can be stably transformed into
31 Nine Mile II and maintained at a similar low copy like QpH1. Importantly,
32 transformation with pQGK cured the endogenous QpH1 due to plasmid
33 incompatibility. Compared to a Nine Mile II transformant of a RSF1010-based vector,
34 the pQGK transformant shows an identical one-step growth curve in axenic media, a
35 similar growth curve in Buffalo green monkey kidney cells, an evident growth defect
36 in macrophage-like THP-1 cells, and dramatically reduced ability of colonizing bone
37 marrow-derived murine macrophages. In the SCID mouse infection model, the pQGK
38 transformants caused a lesser extent of splenomegaly. Moreover, the plasmid biology
39 was investigated by mutagenesis. We found CBUA0037-0039 are essential for
40 plasmid maintenance, and CBUA0037-0038 account for plasmid compatibility. Taken
41 together, our data suggest that QpH1 encodes factor(s) essential for colonizing murine
42 macrophages, and to a lesser extent for colonizing human macrophages. This study
43 highlights a critical role of QpH1 for C. burnetii persistence in rodents, and expands
44 the toolkit for genetic studies in C. burnetii.
45
46 Author summary bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
47 It is postulated that C. burnetii recently evolved from an inherited symbiont of ticks
48 by the acquisition of novel virulence factors. All C. burnetii isolates carry a large
49 plasmid or have a chromosomally integrated plasmid-like sequence. The plasmid is a
50 candidate virulence factor that contributes to C. burnetii evolution. Here we describe
51 the construction of novel shuttle vectors that allow to make plasmid-deficient C.
52 burnetii mutants. With this plasmid-curing approach, we characterized the role of the
53 QpH1 plasmid in in vitro and in vivo C. burnetii infection models. We found that the
54 plasmid plays a critical role for C. burnetii growth in macrophages, especially in
55 murine macrophages, but not in axenic media and BGMK cells. Our work highlights
56 an essential role of the plasmid for the acquisition of colonizing capability in rodents
57 by C. burnetii. This study represents a major step toward unravelling the mystery of
58 the C. burnetii cryptic plasmids.
59
60 Introduction
61 C. burnetii is a Gram-negative intracellular bacterium that causes Q fever, a world
62 widely distributed zoonosis [1]. It is highly infectious and is classified as a potential
63 biowarfare agent [2]. Its infections in humans are mostly asymptomatic but may
64 manifest as an acute disease (mainly as a self-limiting febrile illness, pneumonia, or
65 hepatitis) or as a chronic disease (mainly endocarditis in patients with previous
66 valvulopathy) [1]. All C. burnetii isolates maintain a large cryptic plasmid or
67 plasmid-like chromosomally integrated sequence [3]. This absolute conservation of
68 plasmid sequences suggests they are critical in C. burnetii survival [4]. Increased
69 knowledge of the plasmid sequences will enhance our understanding of C. burnetii
70 biology and pathogenesis and will be important for guiding the design of live bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
71 attenuated vaccine strains for the prevention of Q fever.
72 C. burnetii has at least five different plasmid types: four different plasmids
73 (QpH1, QpRS, QpDV, and QpDG) and one type of QpRS-like chromosomally
74 integrated sequence [5-9]. The plasmids range from 32 to 54 kb in size and share a 25
75 kb core region [3]. Characterization of these plasmid types led to the classification of
76 five associated genomic groups. The hypothesized correlation of plasmid types and
77 genomic groups with the clinical outcomes of Q fever was controversial [3, 10, 11],
78 but the concept of genomic-group specific virulence was supported by mouse and
79 guinea pig infection studies [12, 13]. According to studies on C. burnetii mostly from
80 Europe and North America, this bacterium is considered having a clonal population
81 structure with low genetic diversity [14-16]. Future studies on more C. burnetii
82 isolates especially from other regions will help to understand its global genetic
83 diversity.
84 The sequences of all five representative plasmid types in C. burnetii have been
85 determined [3, 7, 8, 17, 18]. Paul et al. analyzed the diversity of plasmid genes by
86 using DNA microarrays [3]. Nine predicted functional plasmid ORFs are conserved in
87 all plasmid sequences. Two of these ORFs appear to encode proteins similar to phage
88 proteins involved in tail assembly and site-specific recombination, while seven of
89 them encode hypothetical proteins. Three (CBUA0006, -0013 and -0023) of these
90 conserved plasmid hypothetical proteins are secreted effectors of the type IV secretion
91 system [4]. During ectopic expression in HeLa cells, these effectors localize at
92 different subcellular sites, suggesting roles in subversion of host cell functions. In
93 addition, novel plasmid-specific effectors were also identified, supporting the concept
94 of pathotype virulence [19]. Besides the identification of plasmid-encoded secretion
95 effectors, the plasmid is also speculated to be able to be transferred to the host cell bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
96 [20].
97 The advent and foregoing improvement of axenic culture techniques greatly
98 facilitates studies on C. burnetii biology [21-24]. C. burnetii replicates within
99 phagolysosome-like parasitophorous vacuoles (termed Coxiella-containing vacuole,
100 or CCV) in mononuclear phagocytes. Genetic studies revealed that the CCV
101 biogenesis and C. burnetii growth in host cells require a type IV secretion system and
102 a repertoire of type IV effectors [25-28]. The role that these effectors play in C.
103 burnetii pathogenicity is of intense interest. However, regarding the plasmid effectors,
104 reports on their mutants are scarce. By using transposon mutagenesis, Martinez et al.
105 identified mutants of eight QpH1 genes and observed strong phenotypes with mutants
106 of parB and repA, surprisingly, not the mutant of the conserved secretion effector
107 CBUA0023 [29]. Regardless of the identification of plasmid secretion effectors, a role
108 for the plasmid in C. burnetii pathogenesis remains elusive.
109 Since the first development of axenic media, the C. burnetii genetics toolbox has
110 been expanding [30, 31]. Current genetic tools for C. burnetii include, but are not
111 limited to, transposon systems [32], RSF1010 ori-based shuttle vectors [4, 33] and
112 targeted gene inactivation systems [31]. However, there have been no reports of
113 studies using these tools to decipher roles of plasmids in C. burnetii pathogenesis.
114 Here, we describe a set of QpH1-derived shuttle vectors for C. burnetii transformation.
115 These shuttle vectors have different compatibilities with QpH1 and different
116 stabilities in C. burnetii. A QpH1-deficient strain of C. burnetii was constructed by
117 transforming with the pQGK vector. Infection experiments show that QpH1 is
118 essential for colonizing bone marrow-derived murine macrophages. This work
119 increases our knowledge on plasmid biology and provides new genetic tools for
120 investigating C. burnetii pathogenesis. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
121
122 Results
123 C. burnetii plasmid has a putative iteron-like origin of replication
124 The QpH1 plasmid of the Nine Mile isolate is 37,319-bp in size and encodes 40 ORFs,
125 of which only 11 are similar to proteins of known function (Figure 1A) [18]. Besides
126 its cryptic role in pathogenesis, the plasmid’s mechanism of replication and
127 partitioning also lacks investigation. A gene cluster CBUA0036-39 encodes homologs
128 of ParB, ParA, RepB and RepA, respectively, which are supposed to function in
129 plasmid replication and partitioning based on protein similarities. The plasmid was
130 found to have a low copy number [10], but its origin of replication has never been
131 identified. With equicktandem of the EMBOSS software suite [34, 35], we found a
132 putative origin of replication -a region composed of four 21-bp iteron-like sequences
133 between CBUA0036 and -37 (Figure 1B). Unlike the highly conserved iterons in
134 other plasmids, all four iteron-like sequences in QpH1 have base variations from each
135 other. And in currently known C. burnetii plasmids, this putative iteron region is
136 highly conserved. The finding of this putative iteron region suggests genes required
137 for QpH1 replication and partitioning are clustered.
138
139 Construction of incompatible plasmid pQGK and curing of endogenous QpH1
140 from Nine Mile II by transformation
141 The RSF1010-ori based shuttle vectors are routinely used for C. burnetii
142 transformation [31, 33]. We first made a RSF1010-ori based shuttle vector pMMGK
143 for transformation (Figure 2A). An incompatible plasmid pQGK was then constructed
144 as suggested by the clustering of genes (CBUA0036-39) for QpH1 maintenance bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
145 (Figure 2B). The hypothetical CBUA0039a gene was also incorporated in pQGK, as
146 this gene locates close to CBUA0039 and might play unknown roles in plasmid
147 maintenance. The plasmids -pMMGK and pQGK were transformed into Nine Mile II,
148 respectively. After passaging in the presence of kanamycin for six to seven times, the
149 transformants were cloned by using semi-solid ACCM-2 plates and characterized by
150 PCR detection of QpH1 ORFs. All QpH1 ORFs can be detected in NMIIpMMGK, but
151 only the CBUA0036-39a ORFs can be detected in NMIIpQGK (Figure 3). The
152 absence of CBUA001-0034a in NMIIpQGK indicates that the endogenous QpH1 was
153 cured by transformation of pQGK.
154
155 QpH1-deficient C. burnetii has normal growth kinetics in axenic media and
156 BGMK cells
157 The transformants of pMMGK and pQGK -NMIIpMMGK and NMIIpQGK derived
158 from a same Nine Mile II clone. In addition to carrying different shuttle vectors, their
159 major genetic difference is the presence or absence of the endogenous QpH1 plasmid.
160 Both shuttle vectors carry the eGFP cassette, and their clonal homogeneity was
161 inspected by observing eGFP expression. We investigated the potential impact of
162 QpH1 on bacterial growth with these two eGFP-labeled transformants. Their growth
163 kinetics in ACCM-2 media and BGMK cells were assessed by measuring their
164 one-step growth curves (Figure 4A-B). These two transformants display almost
165 identical growth kinetics, suggesting QpH1 is not important for C. burnetii growth in
166 axenic media and BGMK cells.
167 Interestingly, though having similar growth kinetics, these two transformants
168 differ significantly in their expression of eGFP genes (Figure 5). The eGFP genes of
169 these two transformants have identical promoters, but the fluorescence intensity of bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
170 NMIIpMMGK appears to be higher than that of NMIIpQGK. The difference in level
171 of eGFP expression between these two transformants may reflex the difference in
172 gene copy numbers. The copy number of RSF1010-ori based shuttle vector is 3 to 6,
173 while the QpH1 copy number is between one to three. Our data show that QpH1 and
174 pQGK have a similar copy number, suggesting that pQGK retains all genes for QpH1
175 replication and partitioning. The copy number of pMMGK is about two to three times
176 of the copy number of pQGK, which is consistent with their expression levels of
177 eGFP. Overall, our data suggest the copy number of QpH1 and pQGK is under highly
178 stringent control.
179
180 QpH1-deficient C. burnetii almost completely fails to colonize murine bone
181 marrow derived macrophages
182 In vitro, C. burnetii can infect a variety of cell types including epithelial and fibroblast
183 cell lines. However, this bacterium has an infection tropism for mononuclear
184 phagocytes in natural infections [36]. We next characterized the growth of
185 NMIIpMMGK and NMIIpQGK in human macrophage-like THP-1 cells and murine
186 bone marrow-derived macrophages (murine BMDM) (Figure 6), both of which can
187 support robust growth of C. burnetii phase II under normoxic culture conditions
188 [37-39]. Equal MOIs of NMIIpMMGK and NMIIpQGK were used to infect THP-1
189 and murine BMDM cells.
190 In THP-1 cells (Figure 6A), NMIIpMMGK has robust growth at 96 hours
191 post-infection, as shown by the densely packed bacterial clumps and intensive eGFP
192 expressions in its large CCVs. In contrast, NMIIpQGK CCVs are partially filled with
193 bacterial clumps and have lower eGFP expression. This evident lower growth rate of
194 NMIIpQGK is confirmed in the one-step growth curves of these two strains (Figure bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
195 6B). It is noted that the lower eGFP expression of NMIIpQGK in THP-1 is likely due
196 to a combination of lower plasmid copy number and lower growth rate. In addition,
197 we repeatedly measured a minor lower (p<0.05) number of NMIIpQGK at the
198 infection time point of zero, which is the starting time after bacterial entry (Figure 6B).
199 Overall, these THP-1 infection results suggest that QpH1 plays roles in C. burnetii
200 attachment and multiplication in human macrophages.
201 We next compared the growth of NMIIpMMGK and NMIIpQGK in murine
202 BMDMs. In murine BMDMs at 96 hours post-infection, NMIIpMMGK forms normal
203 CCVs that can be easily observed in any microscope field (Figure 6C). This
204 observation is consistent with previous finding of robust growth of C. burnetii phase
205 II in murine BMDMs [38]. Contrarily, NMIIpQGK produces almost zero CCVs. In
206 our repeated experiments, only one NMIIpQGK CCV was ever observed. This rarely
207 observed CCV of NMIIpQGK is shown in Figure 6C. Consistent with microscope
208 observations, no growth yield of NMIIpQGK is detected by qPCR (Figure 6D). It is
209 noted our growth yields of NMIIpMMGK are not optimal when compared to the
210 report of Cockrell et al [38], which is likely due to our modified infection procedures,
211 such as centrifugation at room temperature instead of 37ºC. In addition, comparison
212 of the GE numbers at the starting time point of infection suggested that NMIIpQGK
213 also has slightly reduced attachment efficiency (p<0.05), which is similar with
214 findings in THP-1. Altogether, the murine BMDM infection results suggest that QpH1
215 is essential for C. burnetii survival in murine macrophages.
216
217 Plasmid-less C. burnetii can be isolated by using CBUA0037 or -0038-deleted
218 shuttle vectors
219 The shuttle plasmid pQGK can be stably maintained and has a normal plasmid copy bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
220 number in C. burnetii Nine Mile phase II. We next investigated the plasmid genes for
221 plasmid maintenance in pQGK. A PCR-based deletion mutagenesis approach was
222 adopted to generate specific deletions in each of the five QpH1 ORFs
223 (CBUA0036-0039, -0039a) (Figure 7A) [40]. The deletion regions range from 60% to
224 100% of these ORFs. Primers with unique restriction enzyme sites at their 5´ ends
225 were used for PCR amplification as described in Materials and Methods. After
226 digestion with restriction enzymes, purified PCR products were ligated and
227 transformed into E. coli Trans5α. The desired plasmid construct was confirmed by
228 sequencing. The plasmid knockout vectors are referred to as pQGK-D1, -D2, -D3, D4
229 and -D5, corresponding to deletion mutants of CBUA0036, -0037, -0038, -0039 and
230 -0039a, respectively.
231 Transformations of C. burnetii Nine Mile phase II with these knockout vectors
232 were performed. All knockout vectors contain the eGFP cassette. Their transformants
233 were cultured in kanamycin media and were constantly observed by using
234 fluorescence microscopy. After several passages, transformants were cloned for three
235 times on semi-solid plates. The cloned transformants were then cultured in kanamycin
236 media. The stability of the transformants was assessed by observing eGFP expression.
237 Transformation results are summarized in Table 1. Stable transformants carrying
238 pQGK-D1 and -D5 were isolated (Figure 7B). PCR detection of QpH1 genes indicate
239 that QpH1 was also cured from these transformants. The results of pQGK-D1 and -D5
240 reveal that CBUA0036 and -0039a are not required for stable plasmid maintenance in
241 axenic culture of C. burnetii.
242 Transformation with other three deletion vectors yielded transient or unstable
243 transformants in the presence of antibiotic selection. For the transformation with
244 pQGK-D4, green fluorescence was observed at the first passage after electroporation, bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
245 but failed to expand in the presence of kanamycin selection. The green fluorescence
246 can be repeatedly observed at the first passages in repeated experiments. This
247 transient eGFP expression suggests that pQGK-D4 was introduced into C. burnetii by
248 electroporation but cannot be stably maintained.
249 Transformation with pQGK-D2 and -D3 produced significant bacterial yields in
250 the presence of kanamycin selection. However, clones from kanamycin plates always
251 give a mixture of eGFP-positive and eGFP-negative bacterial clumps. Further
252 identification of these eGFP negative clones revealed that these clones are double
253 deficient of QpH1 and the deletion mutant of pQGK (Figure 7B-C). This unstable
254 eGFP expression and the obtaining of plasmid-deficient C. burnetii suggest that
255 pQGK-D2 and -D3 are incompatible with QpH1 and their transformants are
256 constantly losing their shuttle plasmid due to partitioning deficiency. A
257 plasmid-deficient C. burnetii clone NMIIp- was isolated by three successive cloning
258 of pQGK-D2 transformants. Altogether, these results show that CBUA0037, -0038
259 and -0039 are essential genes for QpH1 maintenance and the pQGK-D2 and -D3
260 vectors are valuable tools for constructing plasmid-deficient C. burnetii.
261 Table 1. Viability and stability of C. burnetii Nine Mile phase II transformed with
262 five pQGK deletion derivatives
Phenotypes of transformants growing in kanamycin media Plasmid Bacterial growth eGFP stability pQGK-D1 + stable (ΔCBUA0036) pQGK-D2 + unstable (ΔCBUA0037) pQGK-D3 + unstable (ΔCBUA0038) pQGK-D4 - transient (ΔCBUA0039) pQGK-D5 + stable (ΔCBUA0039a) 263 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
264 QpH1-deficient C. burnetii has reduced pathogenicity in SCID mice
265 The SCID mouse model was established for identifying virulence factors in C.
266 burnetii phase II [41]. We next analyzed the plasmid’s potential role in C. burnetii
267 pathogenesis by using this model (Figure 8). Four C. burnetii strains -the parent Nine
268 Mile phase II (NMII) and its three derivatives (NMIIpMMGK, NMIIpQGK and
269 NMIIp-) were used to infect SCID mice by peritoneal (IP) injections. Compared to
270 PBS control group, all four C. burnetii strains induced different levels of
271 splenomegaly in infected mice (Figure 8A-B). However, it is apparent that
272 QpH1-bearing strains (NMII and NMIIpMMGK) induced bigger splenomegaly that
273 QpH1-deficient strains (NMIIpQGK and NMIIp-) (p<0.05), suggesting QpH1 plays a
274 role in pathogenicity. It is noted that NMIIpMMGK has a tendency of causing bigger
275 splenomegaly than its parent NMII (p=0.10), indicative of enhanced virulence by the
276 pMMGK plasmid. This enhanced virulence of NMIIpMMGK is likely associated with
277 this strain’s ability of normoxic growth. Characterization of C. burnetii normoxic
278 growth was described in a separate manuscript [42]. Moreover, the sizes of
279 splenomegaly are in accordance with C. burnetii genome equivalents in all infected
280 mice (Figure 8C). It is also obvious that NMIIpQGK retains partial pathogenicity in
281 SCID mice, which is inexplicable as this strain has lost the ability of infecting mouse
282 BMDMs generated from C57BL/6J. Altogether, our data suggest QpH1 plays a partial
283 role in pathogenicity in the SCID mouse model.
284
285 Discussion
286 All C. burnetii has one of the four different types of plasmids or one plasmid-like
287 chromosomally integrated sequence. The plasmids’ role in C. burnetii biology has
288 been implicated by the identification of type IV secretion effectors [4, 19]. Our goal bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
289 was to construct and characterize plasmid-deficient C. burnetii. We show that
290 QpH1-deficient C. burnetii Nine Mile phase II -NMIIpQGK can be constructed by
291 transformation with a QpH1-based shuttle vector pQGK. The NMIIpQGK strain has
292 normal growth in axenic media and BGMK cells, displays partial growth defects in
293 human macrophages and, most strikingly, shows severe infection-deficiency in mouse
294 BMDMs. In the SCID mouse model, the NMIIpQGK strain has partially reduced
295 pathogenicity. We show that CBUA0037, -0038 and -0039 are essential ORFs for
296 QpH1 maintenance, and unmarked QpH1-deficient mutants can be made by
297 transformation with an unstable CBUA0037, or CBUA0038 deletion shuttle vector.
298 Plasmids are commonly classified by incompatibility (Inc) typing [43]. Plasmids
299 that share the same replication mechanisms are incompatible. The Inc type of C.
300 burnetii plasmids remains to be determined. An incompatible plasmid can be used for
301 making plasmid-deficient C. burnetii strains. Our findings that pQGK can cure the
302 endogenous QpH1 by transformation and has a same copy number of QpH1 indicate
303 that pQGK retains an intact QpH1 replicon including genes for plasmid replication,
304 partitioning and copy number control. The pQGK plasmid can be used for Inc typing
305 of C. burnetii plasmids. The pQGK derivatives -pQGK-D2 or -D3 can be used for
306 making unmarked plasmid-deficient mutants. C. burnetii has four types of plasmids.
307 This plasmid curing approach has potential applications for investigating
308 plasmid-type virulence of C. burnetii.
309 Plasmids can maintain their copy numbers by one of the three general classes of
310 negative regulatory systems [44]. Iterons and their cognate replication (Rep) initiator
311 proteins comprise the iteron-involved negative control system [45]. Our results
312 revealed that QpH1 is of low copy number and has atypical iterons. The replication
313 and copy number control of QpH1 is likely regulated through the iterons-Rep bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
314 complex. Iterons are able to exert plasmid incompatibility [46]. Further studies are
315 required to understand the iterons involved regulation of plasmid replication in C.
316 burnetii. In addition, these iterons’ cognate RepA protein -annotated CBUA0039, is
317 supported by our finding of transient transformation with the pQGK-D4 vector. An
318 AT-rich region, locating between the iterons and CBUA0037, could be another
319 essential element for QpH1 replication [47].
320 QpH1 is a plasmid of low copy number [10]. Low-copy plasmids have an active
321 partitioning system. Two putative partitioning proteins -CBUA0036 (ParB) and -0037
322 (ParA), have been annotated based on protein homology. Our results suggest that
323 CBUA0037 and -0038 are essential partitioning proteins while CBUA0036 is
324 nonessential for plasmid maintenance. The commonly used RSF1010-ori based
325 shuttle vectors for C. burnetii is also used in the genetic studies of Legionella
326 pneumophila [33]. Our identification of essential genes for QpH1 maintenance might
327 be useful for constructing QpH1 based shuttle vectors that can be stably maintained in
328 L. pneumophila.
329 Besides genes for plasmid maintenance (replication, copy number control and
330 partitioning), plasmids from pathogenic microbes often contain genes associated with
331 virulence and/or antibiotic resistance [40]. Three of the 40 QpH1 ORFs are essential
332 for plasmid maintenance. Most of other ORFs likely contribute to C. burnetii
333 virulence in various ways. Our finding of QpH1-deficient C. burnetii failing to
334 colonize murine BMDMs likely explains the high conservation of plasmid genes in C.
335 burnetii. We postulate that C. burnetii plasmids are essential for bacterial survival in
336 natural reservoir hosts, including rodents. The role of rodents as a potential reservoir
337 was suggested by finding C. burnetii infections in mice in different areas and
338 countries [48-50]. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
339 How might plasmid genes contribute to the colonization of murine BMDMs?
340 Previous studies using human cells found that the type IV secretion system and a
341 repertoire of type IV effectors required for the CCV biogenesis and C. burnetii growth
342 [25-28]. The role that these type IV effectors play in C. burnetii pathogenicity is of
343 intense interest [51]. Plasmid encoded effectors are possibly involved in the reduced
344 growth efficiency of QpH1-deficient C. burnetii on THP-1 cells, and might also relate
345 to the failed colonization of murine BMDMs by QpH1-deficient C. burnetii. Newton
346 and co-workers found that effector proteins can be translocated as early as 1 h
347 post-infection in murine BMDMs, while effector translocation can only be detected
348 after 8 h post-infection in HeLa cells [52]. We surmise that early translocation of
349 effectors might play an essential role in C. burnetii resisting antimicrobial activities of
350 murine BMDMs. Further studies to investigate the pathogenesis of plasmid genes
351 required for C. burnetii infection and growth in murine BMDMs are warranted.
352 The QpH1-deficient C. burnetii almost completely abrogates its capacity of
353 infecting murine BMDMs. Thus, it is unexpected that this mutant has only partially
354 reduced its pathogenicity in SCID mice. It is possible that the antimicrobial capacity
355 of macrophages is also dampened in this immunodeficiency mouse model. The
356 immunocompetent animal model with C. burnetii phase I as control may be more
357 appropriate for studying plasmid pathogenesis [12]. It is also unexpected that the
358 pMMGK transformant appears to have enhanced pathogenicity. Though similar with
359 other RSF1010-ori based shuttle vectors, the pMMGK vector confers an unexpected
360 capability of normoxic growth on C. burnetii, which likely relates to the
361 transformant’s enhanced virulence. The normoxic growth of C. burnetii was
362 characterized in a separate manuscript [42].
363 In conclusion, we investigated the functions of C. burnetii QpH1 plasmid genes. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
364 We report that QpH1 is a low copy plasmid. We propose that the intergenic region
365 between CBUA0036 and -0037 is the plasmid’s origin of replication, and the
366 plasmid’s copy number is regulated through atypical iterons within this region. The
367 CBUA0037, -0038 and -0039 ORFs encode two partitioning proteins and Rep
368 proteins, respectively. QpH1 can be cured by transformation with pQGK or its two
369 derivatives (pQGK-D2 and -D3). We present evidence that QpH1 is nonessential for
370 C. burnetii growth in axenic media and BGMK cells. QpH1-deficient C. burnetii
371 phase II has minor growth deficiency in human macrophages. Most importantly, we
372 found QpH1 is essential for C. burnetii colonization of murine BMDMs, highlighting
373 an important role of the plasmid for C. burnetii persistence in rodents. It is certain that
374 more plasmid related phenotypes remain to be identified. Our shuttle vectors will
375 further the progress of genetic studies of C. burnetii and its plasmids. This work
376 represents an important step toward unravelling the mystery of plasmid conservation
377 in C. burnetii.
378
379 Methods
380 Bacterial strains, cell lines and key reagents
381 C. burnetii Nine Mile phase II (NM II) is from our laboratory strain collection and
382 was passaged in ACCM-2 as previously described [22]. E. coli Trans5α (TransGen
383 Biotech) was used for recombinant DNA procedures and was grown in Luria-Bertan
384 (LB) media. Buffalo green monkey kidney cells (BGMK) were grown in RPMI 1640
385 medium supplemented with 10% FBS. Human monocytic leukemia cells (THP-1)
386 were cultured in high-glucose-containing Dulbecco’s Modified Eagle Medium
387 (DMEM) supplemented with 10% FBS. Murine bone marrow-derived macrophages
388 (BMDM) were generated from C57BL/6J female mice as described by Cockrell et al bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
389 [38]. Primers used in this study are listed in S1 Table. Escherichia coli Trans5α
390 (TransGen Biotech) was used for all plasmid amplification.
391
392 Construction of a RSF1010 ori-based shuttle vector pMMGK
393 RSF1010 ori-based shuttle vectors are commonly used for C. burnetii transformation
394 [30, 33]. We constructed a RSF1010 ori-based vector pMMGK based on the backbone
395 of pMMB207 (kindly provided by Xuehong Zhang from Shanghai Jiaotong
396 University, Shanghai, China). Promoter regions of CBU0311 and CBU1169 were
397 amplified with primer pairs P311-F/P311-R and P1169- F/P1169-R, respectively. The
398 KanR and eGFP genes were amplified from pEASY-T1 and pEGFP-C1, with primer
399 pairs Kan-F/Kan-R and eGFP-F/eGFP-R, respectively. Overlapping PCR with primers
400 P311-F/Kan-R was used to produce the P311-eGFP-P1169-KanR segment. Two
401 segments -pUC ori and ampicillin resistance gene (AmpR) were amplified from
402 pEASY-T1 with primer pairs pUC-F/pUC-R and Amp-F/Amp-R, respectively.
403 Overlapping PCR with primers P311-F/pUC-R was used to produce the
404 P311-eGFP-P1169-KanR-pUC ori segment. This segment and the AmpR-containing
405 segment were digested with Nhe I and Xho I, and ligated to generate the vector pAGK.
406 The RSF1010 backbone and the P311-eGFP-P1169-KanR segment were amplified
407 from pMMB207 and pAGK, with primer pairs RSF1010-F/RSF1010-R and
408 P311-F/KAN-R, respectively, then were digested with Nhe I and Xho I and ligated to
409 generate the shuttle vector pMMGK (S2 Appendix).
410
411 Construction of C. burnetii QpH1-based shuttle vector pQGK
412 The shuttle vector pQGK was constructed by replacing the AmpR region of pAGK
413 with a predicted region for plasmid replication and partitioning in QpH1. Briefly, a bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
414 5.6-kb segment covering CBUA0036-39a was amplified with primers pQ-F and pQ-R.
415 PCR amplifications were performed in 100-μl reaction volumes containing 50 ng
416 DNA, a 0.2 mM concentration of each dNTP, a 0.2 μM concentration of each primer,
417 2.5 U TransStart FastPfu DNA polymerase (TransGen Biotech), and 20 μl buffer. PCR
418 conditions were as follows: initial denaturation at 95°C for 4 min followed by 2
419 cycles of amplification at 95°C for 30s, 55°C for 30s and 72°C for 1 min every two kb
420 and then 28 cycles of amplification at 95°C 30s, 57°C for 30s and 72°C for 1 min
421 every two kb and a final extension at 72°C for 10 min. This QpH1 segment and the
422 pAGK plasmid were digested with Nhe I and Xho I and ligated to produce the shuttle
423 plasmid pQGK (S3 Appendix).
424
425 Construction of deletion derivatives of pQGK
426 Deletion derivative of pQGK were constructed using a modified PCR-based approach
427 [40]. Primers pairs for knocking out CBUA0036-39a were shown in Table S1 in the
428 supplemental material. Briefly, PCR amplifications were performed using TransStart
429 FastPfu DNA polymerase (TransGen Biotech) and PCR conditions were described as
430 above. PCR products were purified by a QIAquick PCR purification kit (Qiagen) and
431 doubly digested with DMT (to remove template DNA, TransGen Biotech) and
432 restriction enzymes as indicated. Digested PCR products were ligated using Quick T4
433 DNA ligase (NEB) and transformed into E. coli Trans5α. Clones were screened for
434 plasmids containing QpH1 ORFs but lacking the deleted region by PCR.
435
436 Transformation of C. burnetii Nine Mile phase II
437 All plasmids used in this study were purified using a plasmid Maxi Kit (Qiagen) and
438 subjected to sequencing confirmation. The resulting vector was transformed into C. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
439 burnetii Nine Mile phase II by electroporation [31]. For every passage after
440 electroporation, C. burnetii was cultured at 2.5% O2 and 5% CO2 for seven days in
441 ACCM-2 containing 400 μg/mL kanamycin. Fluorescence microscopy was conducted
442 to check eGFP expression after each passage. When eGFP expression was easily
443 observed in bacterial clumps, semi-solid agarose plates were then used for three
444 successive cloning. C. burnetii transformants were expanded in ACCM-2, suspended
445 in SPG (0.25M sucrose, 10mM sodium phosphate, 5mM L-glutamic acid) and kept
446 frozen at -80ºC. Genome copy numbers were determined by Taqman probe qPCR
447 specific to dotA [53].
448
449 PCR identification of transformants
450 The presence or absence of QpH1 in C. burnetii transformants were detected by PCR.
451 Total DNAs were extracted using DNeasy blood & tissue kit (Qiagen), then used as
452 templates for PCR detection with TransTaq high fidelity DNA polymerase (TransGen)
453 and QpH1 gene primers (S4 Table). PCR conditions are as follows: initial
454 denaturation at 94ºC for 5 min, followed by 35 cycles of 94ºC for 30 s, 55ºC for 30 s,
455 72ºC for 90 s.
456
457 One-step growth curves
458 To measure the one-step growth curves in axenic media, seed stocks of C. burnetii
459 was inoculated at a density of 2×106 GE/ml in 96-well plates containing 150 μl
460 ACCM-2 medium per well. Cultures were incubated in a CO-170 tri-gas incubator at
461 37°C with 2.5% O2 and 5% CO2. Samples of axenic culture were collected at
462 indicated time points, then were centrifuged at 20,000 g for 10 min to harvest
463 bacteria. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
464 To measure the one-step growth curves in cells, seed stocks of C. burnetii was
465 plated onto 24-well cell (BGMK, THP-1 and BMDM) monolayers at an MOI of 50.
466 Then, 24-well plates were centrifuged at 800 g for 25 min at room temperature, placed
467 in a CO2 incubator (5% CO2) at 37ºC for 2 hours. Cells were then washed twice with
468 PBS and 1 ml cell medium was added to each well. At indicated time, cell samples
469 were harvested by using trypsin (Hyclone) treatment and centrifugation.
470 Total DNAs were extracted with DNeasy Blood Tissue Kit (Qiagen). Genome
471 copy numbers of C. burnetii in both axenic and cell cultures were determined by
472 Taqman probe qPCR as previously described [53]. PCR conditions were as follows:
473 initial denaturation at 94ºC for 10min, followed by 40 cycles of amplification at 94ºC
474 for 15 s, 60ºC for 1min. All samples were performed in quadruplicate.
475
476 SCID mice infections
477 SCID (CB17/Icr-Prkdcscid/IcrlcoCrl) mice were purchased from Vital River
478 Laboratory Animal Technology Co., Ltd (Beijing, China). Freshly cultured C. burnetii
479 and its transformants –NMIIp-, NMIIpMMGK and NMIIpQGK were diluted to 5×107
480 GE/mL in PBS. Six six-week old female mice per group were infected with 1×107 GE
481 C. burnetii in 200 μl PBS via intra-peritoneal injection. At 18 days postinfection,
482 hearts, lungs, livers, and spleens were aseptically harvested and stored at -80°C,
483 additionally, spleens were weighted to determine splenomegaly (spleen weight/body
484 weight). All samples were individually added up to 2 mL with PBS and homogenized
485 using a glass grinder. DNAs were purified from 20 μL tissue homogenate with
486 DNeasy Blood Tissue Kit (Qiagen) and were used to determine C. burnetii GE by
487 qPCR as described above. All animal experimental procedures involved were
488 performed according to protocols approved by the Institutional Animal Care and Use bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
489 Committee of Fifth Medical Center, General Hospital of Chinese PLA. The project
490 ethics review approval number is IACUC-2018-0020.
491
492 Acknowledgments
493 This work was supported by National Natural Science Foundation of China (No.
494 31570177), Fundamental Research Funds for Central Universities (No.
495 BUCTRC201917), Key Project of Beijing University of Chemical Technology (No.
496 XK1803-06) and Foundation of State Key Laboratory of Pathogen and Biosecurity
497 (No. SKLPBS1409).
498
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703
704 Supporting information
705 S1 Table. Primers for plasmids construction.
706 S2 Appendix. Complete sequence of the pMMGK plasmid.
707 S3 Appendix. Complete sequence of the pQGK plasmid
708 S4 Table. Primers for PCR Identification.
709
710 Figure Legends
711 Figure 1. Map of the QpH1 plasmid (A) and the sequence of its predicted origin
712 of replication, which is composed of four 21-bp iteron-like sequences (B). bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
713 The conserved ORFs (green) [3], predicted ORFs for plasmid maintenance (blue) and
714 predicted ori (red) are as indicated (A). In the iteron alignment (B), dots represent
715 nucleotides that are identical to those in the most upper iteron. The first and last
716 nucleotides in each iteron are given as its position in the complete plasmid.
717
718 Figure 2. Map of two shuttle vectors -pMMGK (A) and pQGK (B).
719 Both vectors share identical KanR and eGFP genes, driven by P1169 and P311
720 promotors, respectively. The inner circle of the pMMGK vector represents the
721 backbone of a RSF1010 ori-based plasmid pMMB207 (A). The inner circle of the
722 pQGK vector represents the fragment from QpH1 (B). The genes and their direction
723 of transcription are represented by the boxes of the outer circle.
724
725 Figure 3. PCR detection of QpH1 ORFs in two transformants -NMIIpMMGK (A)
726 and NMIIpQGK (B).
727 All QpH1 ORFs are present in NMIIpMMGK while only the CBUA0036-0039a
728 ORFs can be detected in NMIIpQGK.
729
730 Figure 4. One-step growth curves of two transformants -NMIIpMMGK (solid line)
731 and NMIIpQGK (dotted line) in axenic media (A) and BGMK cells (B).
732 Growth was quantified by measuring GE. Results of axenic and cell cultures are from
733 six and four separate replicates, respectively.
734
735 Figure 5. Phase and fluorescence microscopy of NMIIpMMGK (A, C) and
736 NMIIpQGK (B, D) in axenic media (A-B) and BGMK cells (C-D).
737 Compared to NMIIpQGK, NMIIpMMGK appears to have higher level of eGFP
738 expression in both axenic media and BGMK cells. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
739
740 Figure 6. Growth of C. burnetii transformants -NMIIpMMGK and NMIIpQGK
741 on macrophage-like THP-1 cells (A, B) and bone marrow-derived murine
742 macrophages (BMDMs) (C, D).
743 (A) In THP-1 cells at 96 hours postinfection, CCVs of NMIIpMMGK have strong
744 eGFP expressions and are filled with bacterial particles while CCVs of NMIIpQGK
745 have weak eGFP expression and have much less bacterial particles. (B) Compared to
746 NMIIpMMGK, NMIIpQGK has lower GEs at the initial attachment and lower growth
747 yields throughout the culture period. (C) In BMDMs, NMIIpMMGK forms
748 normal-sized CCVs and has strong eGFP expression while NMIIpQGK almost
749 completely fails to colonize. A rarely observed CCV of NMIIpQGK is shown. (D) In
750 BMDMs, the GEs of NMIIpMMGK increased close to two logs while the GEs of
751 NMIIpQGK have no significant growth throughout the culture period.
752
753 Figure 7. Construction of five individual QpH1 ORF knockout vectors and PCR
754 identification of their transformants.
755 (A) Schematic representation of deleted regions (white) of pQGK in five individual
756 QpH1 ORF knockout vectors. The coding sequences of CBUA0036-0039a and their
757 direction of transcription are shown by the arrows on the line. (B) The CBUA0006
758 ORF is absent in transformants of pQGK, -D1, -D2, -D3, -D5, suggesting these
759 transformants are QpH1-deficient. The CBUA0039 ORF is also absent in
760 transformants of pQGK-D2 and -D3, suggesting these two transformants contain no
761 any plasmid. (C) The CBUA0036-0039a ORFs are absent in NMIIpQGK-D2 and
762 NMIIpQGK-D3, but present in NMIIpQGK.
763 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
764 Figure 8. Splenomegaly of SCID mouse livers after intra-peritoneal challenge
765 with C. burnetii.
766 SCID mice were challenged with PBS or 1×107 GE of C. burnetii via intra-peritoneal
767 route and sacrificed 18 days after challenge. (A) Spleens were removed from control
768 and challenged mice. (B) Splenomegaly calculated as spleen weight as a percentage
769 of total body weight at the time of necropsy. (C) Genome equivalents calculated using
770 TaqMan real-time PCR with DNA purified from infected spleens from 6 mice on 18
771 days after challenge with C. burnetii. For all panels, error bars represent standard
772 deviations from the mean.
773 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.22.001875; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.