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. Author manuscript; available in PMC: 2025 Nov 26.
Published in final edited form as: Mol Microbiol. 2025 Feb 6;123(3):265–278. doi: 10.1111/mmi.15344

Remote Regulation by VirB, the Transcriptional Anti-Silencer of Shigella Virulence Genes, Provides Mechanistic Information

Cody Cris 1,2, Monika M A Karney 1, Juniper S Rosen 1,3, Alexander D Karabachev 1,4, Elizabeth N Huezo 1, Helen J Wing 1
PMCID: PMC12645490  NIHMSID: NIHMS2124066  PMID: 39912328

Abstract

Classical models of bacterial transcription show regulators binding close to promoter elements to exert their effect. However, the scope for long-range regulation exists, especially by nucleoid structuring proteins, like H-NS. Here, long-range regulation by VirB, a transcriptional regulator that alleviates H-NS-mediated silencing of key virulence genes in Shigella species, is explored in vivo to test the limits of long-range regulation and provide further mechanistic insight. VirB-dependent regulation of the well-characterized icsP promoter persists if its cognate site is repositioned 1 kb, 3.3 kb, and even 4.7 kb further upstream than its native position in a plasmid reporter. VirB-dependent regulation diminishes with binding site distance. While increasing cellular VirB pools elevated promoter activity in all constructs with wild-type VirB binding sites, it did not generate a disproportionate increase in promoter activity from remote sites relative to the native site. Since VirB occludes a constitutively active promoter (PT5) when docked adjacent to its −35 element, we next moved the VirB binding site far outside the promoter region. We discovered that VirB still interfered with promoter activity. These findings and those generated from molecular roadblocks engineered around a distally located VirB-binding site are reconciled with the various models of transcriptional regulation by VirB.

1 |. Introduction

In bacteria, remote regulation is poorly understood. It defies the traditional view of transcriptional control in bacteria, where transcription factors influence the activity of promoters by binding to promoter-proximal (< 250 bp) binding sites (Collado-Vides, Magasanik, and Gralla 1991). Nevertheless, some bona fide examples of remote transcriptional regulators exist (e.g., NtrC family members, λ CI, DeoR and others; Belitsky and Sonenshein 1999; Ninfa, Reitzer, and Magasanik 1987; Reitzer and Magasanik 1986; Ueno-Nishio, Backman, and Magasanik 1983; Ueno-Nishio et al. 1984, Wedel et al. 1990, Dandanell et al. 1987; Dodd et al. 2004; Dunn et al. 1984; Czaplewski et al. 1992; Flashner and Gralla 1988; Narang 2007), revealing the scope for this regulatory control. Furthermore, a high frequency of transcription factor binding sites are located at remote locations relative to annotated promoters (reviewed in Galagan, Lyubetskaya, and Gomes 2013), but the role of these sites in gene regulation remains unclear. Additionally, nucleoid-associated proteins (NAPs), such as histone-like nucleoid-structuring protein (H-NS), exert remote regulatory effects on transcription (Dole, Nagarajavel, and Schnetz 2004; Owen-Hughes et al. 1992; Kalafatis and Slauch 2021) through their organization and compaction of bacterial chromatin, again highlighting the need to better understand remote regulatory processes that govern transcription of bacterial DNA.

In Shigella spp., many genes on the large (220 kb) virulence plasmid are transcriptionally silenced by the chromosomally encoded NAP, H-NS (Beloin and Dorman 2003; Le Gall et al. 2005; Porter and Dorman 1997). This silencing is alleviated by the anti-silencing protein VirB, which is responsible for the upregulation of key virulence plasmid genes (Basta et al. 2013; Turner and Dorman 2007; Wing et al. 2004). Despite controlling the expression of about 50 genes on the large virulence plasmid, either directly or indirectly (Adler et al. 1989; Hall et al. 2022; Watanabe et al. 1990; Haidar-Ahmad et al. 2023), a complete understanding of how VirB functions to antagonize H-NS:DNA complexes remains elusive (Gao et al. 2013; Turner and Dorman 2007; Weatherspoon-Griffin et al. 2018), even though these regulatory processes underpin the virulence of Shigella spp.

VirB is unlike other transcriptional regulators. It is a member of the ParB superfamily, a family of proteins needed for the segregation of DNA molecules (both plasmids and chromosomes) to daughter cells during bacterial cell division (Beloin, McKenna, and Dorman 2002). Canonical members of the ParB family bind CTP as a ligand, which promotes the dimeric form of ParB to adopt a sliding clamp conformation (Soh et al. 2019; Osorio-Valeriano et al. 2019; Jalal, Tran, and Le 2020). This change in conformation causes ParB to disengage from its initial DNA binding site, parS, allowing ParB to spread laterally along the DNA with the DNA helix located in the central lumen of the dimeric protein (Soh et al. 2019, Osorio-Valeriano et al. 2019, Jalal, Tran, and Le 2020). Despite VirB having an entirely different cellular role, recent work shows that VirB also binds CTP (Gerson et al. 2023; Antar and Gruber 2023; Jakob et al. 2024) and this ligand is necessary for its anti-silencing of virulence genes (Gerson et al. 2023). Structural predictions also indicate that VirB dimers likely adopt a sliding clamp conformation on DNA (Gerson et al. 2023; Antar and Gruber 2023; Jakob et al. 2024), like ParB, raising questions about how these DNA-binding activities may offset H-NS-mediated transcriptional silencing.

Arguably, the countervailing roles of H-NS and VirB have been most extensively studied at the icsP locus, which encodes a virulence-associated outer membrane protease (Egile et al. 1997; Shere et al. 1997). The icsP promoter is regulated by H-NS and VirB from remote regulatory regions. Transcriptional silencing by H-NS relies on DNA sequences located between 900 and 436 bp upstream of the primary transcription start site (TSS; Hensley et al. 2011, Weatherspoon-Griffin et al. 2018). The silencing mediated by H-NS is alleviated by VirB engaging a remote cis-acting site centered 1137 bp upstream of the TSS and, hence, 237 bp upstream of the region required for H-NS-mediated repression (Castellanos et al. 2009). In backgrounds lacking H-NS, VirB has little to no regulatory effect (Wing et al. 2004).

Three activities ascribed to VirB have been proposed to be involved in its transcriptional anti-silencing activity: spreading, bridging, and modulation of DNA supercoiling (Weatherspoon-Griffin et al. 2018; Socea, Bowman, and Wing 2021; Picker et al. 2023). These activities may function independently, or in some combination, to mechanistically trigger anti-silencing by VirB. The spreading of VirB on DNA was implicated in transcriptional anti-silencing by VirB at the icsP promoter when a molecular roadblock (LacI) positioned between the VirB binding site and the region occupied by H-NS prevented transcriptional anti-silencing (the roadblock had no effect when positioned on the other side of the VirB binding site; Weatherspoon-Griffin et al. 2018). Bridging of VirB is supported by subcellular localization experiments that reveal regulatory active VirB forms discrete fluorescent foci in vivo when translationally fused to green fluorescent protein (GFP; Socea, Bowman, and Wing 2021). These foci are reminiscent of the highly concentrated ParB clusters that assemble partition complexes via a network of stochastic, dynamic ParB interactions that bridge and condense DNA (Broedersz et al. 2014; Sanchez et al. 2015; Tisma et al. 2023). These observations suggest that VirB-GFP foci form through similar VirB-VirB and VirB-DNA interactions and complexes, and these may influence gene regulation.

More recently, DNA topology/supercoiling changes were implicated in VirB-mediated transcriptional control of the icsP promoter (Picker et al. 2023). Topoisomeric changes in plasmid DNA were found to be mediated by VirB-DNA interactions, causing a loss of negative supercoils in vivo and a gain of positive supercoils in relaxed DNA in vitro (Picker et al. 2023). This activity was curious as VirB is not a topoisomerase (Beloin, McKenna, and Dorman 2002). Furthermore, the VirB-mediated loss of negative supercoils was not driven by (i) topoisomerase I, (ii) VirB-dependent transcription, or (iii) H-NS, as it was seen to occur in an hns mutant. Through parallel investigations in cells lacking VirB, a localized loss of negative supercoils in the icsP promoter region was found to relieve transcriptional silencing mediated by H-NS, likely through the remodeling of the H-NS-DNA complex (Picker et al. 2023). This observation led to the current model of transcriptional anti-silencing by VirB, whereby VirB engages and subsequently spreads along DNA, leading to a transient and localized loss of negative DNA supercoils, which, when occurring in the vicinity of H-NS-DNA complexes, alleviates transcriptional silencing mediated by H-NS.

Since the H-NS regulatory region and VirB binding site found at the icsP locus do not have strict spacing requirements or exhibit face-of-the-helix dependency (Weatherspoon-Griffin et al. 2018), in this work, we sought to further enhance our understanding of the mechanism underlying VirB-mediated transcriptional anti-silencing by exploring the potential for VirB to counter H-NS-mediated silencing from remote sites. To do this, we altered the location of the VirB binding site on a variety of lacZ reporter plasmids, thereby increasing the distance between the VirB binding site and the H-NS regulatory region, and tested the VirB-dependent regulatory effect in vivo. These genetic tools also allowed us to assess whether the potency of VirB-mediated anti-silencing diminishes with binding site distance or protein concentration. We reasoned that these experiments would extend support for either VirB spreading or VirB bridging in the mechanism of transcriptional control, as bridging would likely be less impacted by binding site position or cellular pools of VirB. Moreover, these studies may reveal that another protein, VirB, is capable of long-range gene regulation, a phenomenon that is poorly understood and often overlooked in bacteria.

2 |. Results

2.1 |. VirB Regulates the icsP Promoter When Its Binding Site Is Repositioned 1 kb Further Upstream

Previous analysis of the H-NS-regulated virulence gene icsP found that VirB-mediated regulation of its promoter (PicsP) was not affected by deletion of 5, 10, or 50 base pairs (bp) between the VirB binding site (Castellanos et al. 2009; natively centered −1137 relative to the primary TSS, (Hensley et al. 2011)) and the region required for H-NS-mediated silencing located between −900 and −436 bp (Weatherspoon-Griffin et al. 2018). This demonstrated a degree of spatial and phasic flexibility to the remotely located VirB binding site of the icsP promoter and transcriptional regulation by VirB. However, it was unclear if VirB could offset H-NS-mediated silencing of the icsP promoter from a binding site repositioned further upstream. To address this, we introduced a 1 kb DNA fragment into our PicsP-lacZ transcriptional reporter plasmid (WT PicsP; Basta et al. 2013) between the established VirB binding site and region required for silencing by H-NS (Figure 1A). Since the virulence plasmid is known to have high AT content, we sourced the 1 kb sequence from the icsA coding region to match the AT richness of the icsP locus. The resulting reporter plasmid, PicsP+1 kb, possesses a sole VirB binding site 2137 bp upstream of the principal TSS of the icsP promoter (Hensley et al. 2011). The activity of the icsP promoter was measured using β-galactosidase assays in the wild-type S. flexneri 2a strain 2457T and an isogenic virB mutant strain (AWY3). As shown previously (Castellanos et al. 2009), WT PicsP exhibited a VirB-dependent increase in promoter activity, whereas mutating the VirB binding site abolished regulation (mutated PicsP) (Figure 1B). Surprisingly, PicsP+1 kb still exhibited a VirB-dependent effect on icsP promoter activity at endogenous levels of VirB, although the VirB-dependent effect was significantly reduced compared to WT PicsP (Figure 1B; 57.5% ± 7.8% that of WT PicsP). This demonstrates that VirB can relieve H-NS-mediated silencing of the icsP promoter from 1 kb further upstream than its native position (−1137) and raises the possibility that the distance of the VirB binding site from the icsP promoter is inversely related to the VirB-dependent regulatory effect.

FIGURE 1 |.

FIGURE 1 |

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VirB can regulate the icsP promoter from 1 kb further upstream. (A) Schematic of the icsP promoter region (thick black line) from the PicsP-lacZ reporter plasmid (not drawn to scale); positions of regulatory elements, the VirB binding site (5′-ATTTCAGtATGAAAT-3′; divergent arrows), and the 1 kb DNA insert are shown relative to the position of the principal TSS (+1) (Castellanos et al. 2009; Weatherspoon-Griffin et al. 2018; Hensley et al. 2011). (B) Activity of the icsP promoter for wild-type (WT PicsP), with a mutated VirB binding site (5′-GCCCAGCtCGACCCG-3′; mutated PicsP), or with a 1 kb DNA insert (PicsP+1 kb) in wild-type (2457T) or virB mutant (AWY3) strains of S. flexneri measured using β-galactosidase assays; a representative trial is shown (n = 3). Error bars represent standard deviations. A two-way ANOVA with post hoc Šidák was used to assess statistical significance: *p < 0.05, “ns” = not significant. Full statistical analysis is provided in Table S3.

2.2 |. VirB Can Regulate the icsP Promoter From 4.7 kb Away, and Regulation Is Inversely Related to Binding Site Distance

After observing the clear ability of VirB to abrogate H-NS-mediated silencing of the icsP promoter from a binding site > 2 kb upstream of the promoter, we sought to probe the limits of remote regulation by VirB but with a different approach. In our previous dataset (Figure 1B), we noticed that promoter activity of the PicsP+1 kb reporter was elevated in the absence of VirB, relative to the WT PicsP (Figure 1B; compare light gray bars), suggesting the 1 kb DNA insert elicited moderate promoter derepression, possibly by destabilizing the adjacent H-NS-DNA complex. Therefore, to further probe the limit of remote regulation by VirB without disrupting the icsP promoter region with DNA inserts, we chose to relocate a 225 bp region containing the VirB binding site around the PicsP-lacZ transcriptional reporter plasmid in the plasmid backbone. First, we constructed a reporter series possessing either a wild-type or mutated VirB binding site at both the native position within the icsP promoter region and centered at a position 3317 bp upstream of the TSS (Figure 2A), designated the “medial” position. Separately, we also generated a reporter series with either a wild-type or a mutated VirB binding site at both the native position or on the opposite side of the plasmid, centered 4759 bp upstream of the TSS (Figure 2A), designated the “distal” position. As such, the resulting plasmids (Figure 2A) carried either a single wild-type VirB binding site and a mutated site or two mutated sites, which served as a negative control. These reporters were then assayed for β-galactosidase activity in wild-type and virB mutant backgrounds of S. flexneri.

FIGURE 2 |.

FIGURE 2 |

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VirB can regulate the promoter of icsP from over 4.7 kb away. (A) Schematics of the two series of PicsP-lacZ reporter plasmids. The “medial site” series possesses the wild-type (WT) or mutated (mut) VirB binding site (striped divergent arrow) (Castellanos et al. 2009) at the native position in the promoter region (−1137) and the “medial” position (−3317). The “distal site” series possesses the WT or mutated VirB binding site at the native and the “distal” position (−4759). Medial and distal sites were transplanted on a 225 bp sequence (thick black line) that naturally surrounds the native site in PicsP. (B) Activity of the icsP promoter for the PicsP-lacZ reporters possessing wild-type (WT) or mutated (mut) VirB binding sites at the positions indicated below the graph; reporters were carried by wild-type (2457T) or virB mutant (AWY3) strains of S. flexneri and measured using β-galactosidase assays; a representative trial is shown (n = 3). Error bars represent standard deviations. A two-way ANOVA with post hoc Šidák was used to assess statistical significance: *p < 0.05, “ns” = not significant. Full statistical analysis is provided in Table S4.

As expected, in the presence of VirB, icsP promoter activity was enhanced for the two reporters possessing a wild-type VirB binding site at the native position but was not enhanced for the two reporters with two mutated VirB binding sites (Figure 2B). Remarkably, VirB-dependent upregulation of the promoter was retained in constructs with the wild-type VirB binding site relocated to either 3317 bp or 4759 bp upstream of the TSS, but the VirB-dependent effect was significantly lower; 36.9% ± 3.6% at the medial position and only 11.8% ± 1.4% at the distal position (relative to the wild-type site at its native location; Figure 2B).

Overall, these data demonstrate that VirB can anti-silence the icsP promoter from over 4.7 kb away and confirm the inverse relationship between the distance of the VirB binding site and the regulatory activity of VirB, findings consistent with VirB spreading along the DNA from its initial binding site into the regulatory region of PicsP to elicit its regulatory effect.

2.3 |. Increasing the amount of VirB increases VirB-dependent regulation, but does not lead to a disproportionate increase in regulation from remote sites

Next, we examined how modulating cellular VirB pools affects VirB-dependent regulation of reporter constructs carrying remotely positioned sites. To do this, an l-arabinose-inducible virB expression vector pBAD-virB was introduced into the virB mutant strain AWY3 so that cellular VirB levels could be titrated based on the concentration of the l-arabinose inducer. Using varying concentrations of the inducer (0%, 0.005%, 0.01%, 0.02%, or 0.2% l-arabinose), we first confirmed that cellular VirB levels generated by these inducer concentrations mimicked or exceeded those in wild-type 2457T (Figure 3A,B). Next, the reporters from the PicsP+1 kb, medial, and distal series were assayed in AWY3 pBAD-virB induced with the same titration scheme (0%, 0.005%, 0.01%, 0.02%, or 0.2% l-arabinose). As expected, regulation by VirB was observed in all reporters possessing a wild-type VirB binding site. Again, an inverse relationship between the distance of the VirB binding site and the transcriptional anti-silencing activity of VirB was observed (Figure 3C–E), but, in these constructs, VirB-dependent regulation was enhanced by increasing the cellular VirB pool.

FIGURE 3 |.

FIGURE 3 |

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VirB-mediated regulation increases with elevating cellular VirB pools, but a disproportionate increase from remote sites is not observed. (A) Levels of VirB (35.4 kDa) in the virB mutant strain of S. flexneri (AWY3) as visualized by a western blot. Cells carrying either pBAD-virB or empty pBAD were induced with the indicated concentration of l-arabinose (% l-ara). Native VirB levels in the wild-type strain, 2457T, are shown for comparison. A representative blot is shown. (B) Densitometry of western analyses (n = 2) showing relative VirB levels for native (2457T) and induced AWY3 pBAD-virB. Asterisks represent data that are statistically different from 2457T as determined by two-tailed Student’s t-test (*p < 0.05). (C–E) Promoter activities of (C) PicsP+1 kb, (E) PicsP from the medial site (−3317), and (G) PicsP from the distal site (−4759) in the virB mutant strain of S. flexneri (AWY3) carrying pBAD-virB or empty pBAD and induced with the indicated concentration of l-arabinose. A representative trial is shown (n = 3). Full statistical analysis is provided in Tables S5–S7. A two-way ANOVA with post hoc Šidák was used to assess statistical significance (*p < 0.05). (F–H) Average relative VirB-dependent activity compared to the native icsP promoter at each inducer concentration was calculated from the data represented by panels C–E for the upregulation of (F) PicsP+1 kb, (G) PicsP from the medial site, and (H) PicsP from the distal site. Relative activity across three independent trials (n = 3; each with three biological replicates) was averaged for each experiment; bars represent standard deviations. A one-way ANOVA with post hoc Bonferroni was used to assess statistical significance (p < 0.05) in panels (F–H); the same letters indicate no statistical difference.

Using these data, we could now determine if the increased pool of VirB had disproportionately increased PicsP promoter activity in the PicsP+1 kb, medial, and distal constructs over that generated from the native site in PicsP. We reasoned that this might be the case if transcriptional anti-silencing of PicsP by VirB was mediated by VirB spreading along the DNA towards the H-NS regulatory region in PicsP. After background subtraction (i.e., no inducer), VirB-dependent enhancement of the construct bearing the native site was normalized to 100% at each inducer concentration, and then relative VirB-dependent activity of PicsP in the PicsP+1 kb (Figure 3F), medial (Figure 3G), and distal (Figure 3H) series was expressed as a percentage (formulas provided on respective figure panels). Data across all three trials were compared, allowing an average relative VirB-dependent activity of constructs bearing a remote VirB binding site to be compared to that generated from the native site at each inducer concentration. The results revealed that the average relative VirB-dependent effect from the remote sites remained similar to the VirB-dependent effect from the native site in WT PicsP regardless of the level of virB induction holding at ~60% for PicsP+1 kb, ~58% for the medial site, and, ~33% for the distal site (Figure 3F–H). Thus, even though our titration experiments recapitulate the distance-dependent effect observed at physiological VirB levels, they reveal that remote regulation by VirB of PicsP+1 kb and constructs with the medial and distal sites is not disproportionately enhanced by super-physiological levels of VirB; that is, the remote position of the VirB binding site cannot be compensated by increased VirB cellular pools.

2.4 |. A Molecular Roadblock Can Unidirectionally Abolish VirB-Mediated Regulation of the icsP Promoter When the VirB Binding Site Is Repositioned 4.7 kb Away

In previous work, unidirectional spreading of VirB from its binding site towards the region occupied by H-NS had been implicated as a requirement for transcriptional anti-silencing in vivo (Weatherspoon-Griffin et al. 2018). Given that the VirB binding site of the “distal site” reporter is essentially equidistant from the icsP promoter in either direction (4759 bp upstream versus 4706 bp downstream from the TSS), we wondered whether the remote regulation by VirB from the opposite side of the plasmid could be attributed to VirB spreading along DNA in one direction (through the cat gene), the other direction (through the ori and lacZ regions), or both.

To address this, we employed the lac operon repressor (LacI) as a molecular roadblock to obstruct the spreading of VirB laterally from its binding site at the distal position, using an experimental design similar to that used previously (Weatherspoon-Griffin et al. 2018). Briefly, duplicate LacI recognition sites (lacO1 sites) were engineered to be centered either 100 bp upstream or downstream of the VirB binding site centered at −4759 bp relative to the principal TSS of the icsP promoter (Figure 4A). A medium copy lacIq expression plasmid (pQE2) ensured LacI production, and the association or dissociation of LacI to the lacO sites was controlled by the absence or presence of IPTG, respectively.

FIGURE 4 |.

FIGURE 4 |

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VirB-mediated regulation of PicsP from a binding site more than 4.7 kb away is disrupted by a molecular roadblock docked downstream, but not upstream, of its binding site. (A) Schematics depicting constructs used in the “roadblocking experiments.” Each plasmid has a 225 bp sequence (thick black line) including the VirB binding site from PicsP (divergent arrows) centered at the distal position (−4759 from the primary TSS of the icsP promoter). Tandem lacO sites are centered 100 bp either downstream (top) or upstream (bottom) of the center of the VirB binding site. Double forward slashes indicate undepicted intervening regions. (B) Activity of the icsP promoter from the distal VirB site alone or with tandem lacO sites either downstream (D’stream) or upstream (U’stream); a promoterless reporter (P’less lacZ) was used as a control. Assays were performed in wild-type (2457T) and virB mutant (AWY3) backgrounds of S. flexneri carrying a lacIq expression vector (pQE2; note all Shigella strains naturally lack lacI) and grown in the absence (−) or presence (+) of IPTG. A representative trial is shown (n = 3). Error bars represent standard deviations. A three-way ANOVA with post hoc Šidák was used to assess statistical significance (*p < 0.001). Full statistical analysis is provided in Table S8.

β-Galactosidase activities in the virB mutant background were not influenced by the presence or absence of IPTG (Figure 4B). As expected, in wild-type S. flexneri, the presence or absence of IPTG did not significantly impact icsP promoter activity in the original “distal site only” PicsP-lacZ reporter, because this construct lacks the engineered lacO recognition sites. PicsP activity was, however, diminished under conditions conducive for LacI binding (no IPTG) when the lacO sites were positioned downstream (D’stream lacO) of the VirB binding site, consistent with LacI perturbing VirB spreading toward the 5′ region of the icsP promoter (Figure 4B). In contrast, when the lacO sites were positioned upstream (U’stream lacO) of the VirB binding site, the presence or absence of IPTG did not lead to a change in icsP promoter activity. Thus, even though the VirB binding site is located on the opposite side of the reporter plasmid relative to PicsP, VirB-dependent regulation was only impacted when the LacI roadblock was positioned downstream of the VirB recognition site. Overall, these data suggest that VirB-dependent transcriptional anti-silencing from the distal site is likely attributable to VirB spreading through the cat gene of the plasmid toward the 5′ end of the icsP promoter.

2.5 |. VirB Can Inhibit Transcription Initiation From Sites Located Far Outside of the RNA Polymerase Footprint

To further explore VirB-dependent regulation from remote sites in our small plasmid reporters, we next chose to extend earlier work that had shown VirB blocks the activity of the constitutively active promoter, PT5, when bound immediately adjacent to its −35 element (Karney et al. 2019). As described previously (Karney et al. 2019), pBT-WT carries a VirB binding site centered at −53, relative to the TSS, which places it within the region occupied by RNA polymerase during normal transcription initiation (verified by DNase I protection assay; Karney et al. 2019). Here, we moved the VirB binding site further upstream, outside of the region occupied by RNA polymerase, centering the wild-type or mutated site at either −139 (pRBT series) or −1139 (pRBT+1 kb series) (Figure 5A). We reasoned that if VirB spreads along DNA, regardless of where the VirB binding site is positioned, the PT5 promoter would be occluded in the presence of VirB, and little to no PT5 activity would be detected. Whereas if VirB was forming intra- or inter-plasmid bridges, these would occur at random DNA sites and be unlikely to prevent RNA polymerase from engaging and transcribing the constitutively active T5 promoter.

FIGURE 5 |.

FIGURE 5 |

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VirB hinders PT5 activity when the VirB binding site is located over 1 kb upstream of the transcription start site, regardless of cellular VirB pools. (A) Schematic of the pBT plasmid series. The binding site (striped box) is either a wild-type VirB binding site or a mutated site (Castellanos et al. 2009). Black boxes represent the −10 and −35 promoter elements. In pBT (binding tool), the site is centered at −53 relative to the TSS of PT5; brackets indicate the characterized RNA polymerase (RNAP) binding region in this promoter (Karney et al. 2019). In pRBT (remote binding tool) and pRBT+1 kb (remote binding tool with a 1 kb insert), the site is centered at −139 and −1139, respectively, and is found on a 225 bp sequence (black line) that naturally surrounds the native site in PicsP. (B) PT5 promoter activity associated with the pBT series in the presence (pBAD-virB) or absence (pBAD) of VirB in a S. flexneri virB mutant (Shigella strains naturally lack lacI), as determined by β-galactosidase assays. A representative trial is shown (n = 3). A two-tailed Student’s t-test was used to determine the significance between the presence and absence of VirB for each construct (*p < 0.001). (C) Average relative VirB-dependent hindrance of PT5 in pRBT+1 kb compared to pBT, at each inducer concentration, was calculated from data represented by Figure S1 (n = 2; each with three biological replicates). Relative activity was calculated using the formula provided, and the average was taken. A one-way ANOVA with post hoc Bonferroni was used to assess significance (*p < 0.05); the same letters indicate no statistical difference. A representative trial is shown in Figure S1 and full statistical analysis is provided in Table S9.

To test these ideas, promoter activity of pBT, pRBT, and pRBT+1 kb was measured in the virB mutant, AWY3, carrying either an l-arabinose-inducible virB expression vector pBAD-virB or an empty plasmid control with 0.2% l-arabinose inducer. Regardless of how far upstream the VirB binding site was located, VirB still interfered with the activity of PT5, and to an equivalent extent (Figure 5B). As expected, this interference was not seen when the mutated site was introduced at each of these positions. Thus, the engagement of DNA by VirB can, in some genetic contexts, silence promoters located in the immediate vicinity, as seen for ParB (Rodionov, Lobocka, and Yarmolinsky 1999). Our findings are consistent with VirB spreading along the DNA from its cognate binding site to mediate the silencing of the PT5.

Admittedly, the level of VirB in these experiments was likely above physiological levels, so we next assessed whether lower levels of VirB impact VirB-mediated interference of PT5 activity. To do this, the constructs were assayed in the virB mutant AWY3 carrying pBAD-virB, and virB expression was induced using the following inducer concentrations (0.2%, 0.02%, 0.01%, or 0.005% l-arabinose). These assays revealed similar levels of VirB-dependent interference regardless of the amount of VirB in the system (Figure 5C and Figure S1). Thus, these data show that VirB interferes with promoter activity when its cognate DNA binding site is located far outside of the region occupied by RNA polymerase during transcription initiation and that this interference occurs equivalently at all levels of VirB generated in our titration assays.

To conclude, this study demonstrates the extent of long-range regulation by VirB in vivo, which can occur either by transcriptional anti-silencing or promoter repression. Mechanistically, these regulatory effects are most likely triggered by a small number of VirB dimers spreading along DNA towards the promoter to influence its activity, rather than the VirB dimers forming a contiguous filament on DNA. This is supported by our observations that increasing VirB pools do not disproportionately increase the regulatory effect from remote sites when compared to the natively positioned site and that a molecular roadblock flanking the distal VirB binding site on the side closest to the H-NS regulatory region hinders VirB-dependent regulation of PicsP.

3 |. Discussion

Long-range transcriptional regulation is frequently overlooked and poorly understood in the context of bacterial genomes. Genome-scale technologies, such as ChIP-seq and ChIP-chip, often identify transcription factor binding sites where the transcription factor has no discernable direct local effect on gene expression. To illustrate this, a recent review (Mejia-Almonte et al. 2020) reported that only 25% of 3973 transcription factor-DNA interactions in E. coli were found to regulate local gene expression, and similar observations have been made in many other diverse bacterial genomes. Why do these “orphaned” protein binding sites exist, and how do they function? One possibility is that some of these sites may be used to regulate gene expression remotely. This is especially tantalizing when the protein docking to these sites is capable of modulating or influencing the regional topology and accessibility of DNA. In this work, we investigated the potential for long-range gene regulation by one such protein, the Shigella virulence plasmid-borne transcriptional regulator, VirB.

First, we focused on the characterized activity of VirB as a transcriptional anti-silencer. We found that VirB can counteract H-NS-mediated silencing of the icsP promoter when its cognate site is relocated as far as 4.7 kb away on a 9.5 kb reporter plasmid (Figures 1 and 2). This is remarkable for a bacterial gene regulator. By positioning the cognate VirB binding site at increasing distances from the promoter (Figures 1 and 2), we observed that the VirB-dependent regulatory effect diminished with increasing distance. These data are consistent with our earlier work, which suggested that VirB functions as an anti-silencing protein by spreading along the DNA into the H-NS regulatory region (Weatherspoon-Griffin et al. 2018).

As expected, an increase in promoter activity was observed for all constructs with a wild-type VirB binding site when cellular levels of VirB were increased above native levels in our titration experiments (Figure 3A,B). However, unexpectedly, with all three non-native remote binding site distances tested, the relative VirB-dependent activity from remote sites compared to the native site remained constant (Figure 3F–H), suggesting that the increased VirB pool could not compensate for the increased distance. These data challenge the model of transcriptional anti-silencing where VirB spreads along the DNA to form a contiguous filament between the VirB binding site and the H-NS regulatory region. Instead, our data align better with either a smaller number of VirB dimers spreading along the DNA or VirB bridging upregulating the icsP promoter. Bridge formation by VirB, however, would either require a second VirB binding site, which, to the best of our knowledge, does not exist in our reporters, or, based on ParB literature, hydrolysis of the CTP ligand, which in vitro, at least, promotes bridge formation without the need for a second binding site (Tisma et al. 2022). The CTP hydrolysis rates of VirB, however, are much lower than those of ParB (Antar and Gruber 2023; Jakob et al. 2024), leading us to favor a spreading model where a small number of VirB dimers spread along the DNA to up-regulate the icsP promoter (spreading of ParB family members has been shown to occur without CTP hydrolysis; Balaguer et al. 2021).

To further investigate whether spreading or bridging was more likely, we next chose to test whether VirB-dependent regulation of PicsP-lacZ generated from the distal DNA binding site could be hindered by a molecular roadblock positioned between the distal site and the 5′ region of the icsP promoter (Figure 4). It could, and without evidence of plasmid maintenance issues resulting from VirB interfering with the ori of the reporter plasmids (Figure 4B and data not shown). Moreover, the LacI roadblock on the upstream flank of the VirB binding site had no significant effect on promoter activity when it engaged its site. These data were challenging to reconcile with a model of anti-silencing mediated by VirB bridge formation because a molecular roadblock would be unlikely to impede bridge formation, irrespective of its position. Instead, they favor a model where VirB spreads along the DNA from the VirB binding site into the region occupied by H-NS to trigger VirB-dependent transcriptional anti-silencing.

A final line of support for the spreading activity of VirB on DNA comes from our experiments that show VirB is capable of blocking transcription from a constitutively active semi-synthetic promoter, PT5, even though the VirB binding site is located well outside the region normally occupied by RNA polymerase during transcription initiation (Karney et al. 2019; either 139 bp or 1 kb upstream of the TSS in pRBT and pRBT-1 kb; Figure 5B). Again, in this study, we found that the relative regulatory VirB-dependent effect (promoter repression in this case, not anti-silencing), from remote sites compared to the native site, remained constant regardless of VirB titration, suggesting that a limited number of VirB molecules are needed to repress PT5.

Based on preceding studies, our model of VirB-dependent transcriptional anti-silencing relies upon VirB binding to its site and spreading along the DNA helix toward the region bound by H-NS. This work adds that rather than VirB covering the entire intervening region as a contiguous filament, a smaller number of VirB molecules are needed to spread along the DNA to generate VirB’s regulatory effect, whether that regulatory effect is to relieve the effects of transcriptional silencing by H-NS or to repress a constitutively active promoter like PT5. While it is highly likely that VirB spreads bidirectionally, like ParB (Rodionov, Lobocka, and Yarmolinsky 1999), in the PicsP-lacZ promoter series, we found no evidence that VirB spreading along DNA impacts plasmid maintenance (plasmid levels were similar in the presence and absence of VirB; data not shown), even though the distance between the center of the ori and the distal VirB binding site is only 600 bp. This raises the possibility that a feature of the 225 bp transplanted DNA, which contains the VirB binding site, promotes the directionally biased spread of VirB in vivo. These ideas are currently being investigated. Regardless of whether VirB spreads bidirectionally or not, based on Picker et al. 2023, we favor a model where VirB spreading into a region occupied by H-NS relieves the silencing mediated by this NAP, likely by modulating the local state of DNA supercoiling (Picker et al. 2023).

While this study lends additional support to a model where VirB spreading along DNA is a critical component of transcriptional anti-silencing by VirB, it remains unclear if the genetic context of the regulatory region impacts the VirB-dependent regulatory effect. For instance, the spread of VirB on DNA, and hence transcriptional anti-silencing, may be negatively affected by collisions with transcriptionally active RNA polymerase or genetic loops or domains formed by other DNA-binding proteins. Work addressing these questions is underway and was made possible by the small plasmid reporters described within this study. Additionally, it will be important to study VirB-dependent regulation in the context of the large virulence plasmid of Shigella, where VirB controls the expression of key virulence genes. These experiments may determine the genetic range over which VirB can antagonize H-NS-mediated silencing in its natural genetic context and allow other important questions to be answered in this setting. For instance, does VirB regulate multiple distant promoters simultaneously on the virulence plasmid from a single DNA binding site? What is the genomic distance over which VirB can spread along DNA? Do VirB-DNA interactions ever block the transcription of genes on this plasmid? Surprisingly, a complete understanding of where VirB binds to the Shigella virulence plasmid is currently not known. Consequently, chromatin immunoprecipitation (ChIP)-based experiments that probe VirB-DNA interactions, in combination with RNA-seq analyses carefully designed to characterize the VirB regulon, will likely yield the answers to these questions.

VirB requires the ligand CTP to engage its DNA recognition site (Gerson et al. 2023). Based on work with ParB (Tisma et al. 2022), CTP hydrolysis is likely to release VirB from the DNA. Yet CTP hydrolysis rates by VirB are much lower than for other ParB proteins (Jakob et al. 2024; Antar and Gruber 2023). As such, it remains unclear how the low hydrolysis rate of CTP impacts VirB spread on DNA or transcriptional anti-silencing by VirB. Once VirB engages its DNA binding site on plasmid DNA, a loss of negative supercoils is observed, both in vivo and in vitro (Picker et al. 2023). Since a loss in negative supercoils is sufficient to relieve H-NS-mediated silencing (Picker et al. 2023), it would be interesting to know if VirB spreading triggers these topological changes and if these changes recruit topoisomerases to regions proximal to the VirB-DNA complex. These ideas are framing ongoing research in our laboratory.

In conclusion, VirB is a virulence gene anti-silencer in Shigella that belongs to a novel class of transcriptional regulators. Transcriptional regulation mediated by VirB is facilitated through its engagement of the VirB binding site and the ensuing spread of VirB along the DNA helix, which, in the context of anti-silencing, facilitates transient conformational changes in DNA that elicit alleviation of H-NS-mediated silencing. At this stage, our collective studies on VirB (Castellanos et al. 2009; McKenna and Wing 2020; Weatherspoon-Griffin et al. 2018; Picker et al. 2023; Gerson et al. 2023), support that this co-opted protein from the ParB superfamily (Turner and Dorman 2007; Watanabe et al. 1990; Beloin, McKenna, and Dorman 2002) is not just a transcriptional regulator of Shigella virulence genes, but likely a topological remodeler of DNA to which it binds. Our work also raises questions about other transcriptional anti-silencers (e.g., Ler, LeuO, RovA, SlyA, and AraC-related proteins), a cohort of diverse and un-related DNA-binding proteins that counteract the effects of NAPs, like H-NS, enabling the emergence of novel regulatory circuits (Stoebel, Free, and Dorman 2008). It will be interesting to determine whether these transcriptional regulators also act over large genomic distances and/or mediate changes in DNA topology. Further studies are needed to expand our understanding of the molecular underpinnings of transcriptional silencing and anti-silencing, the interplay of their mediators, and their impacts on DNA topology. This is especially important because these key regulatory processes are central to the transcriptional programs and are integral to bacterial physiology and pathogenesis.

4 |. Materials and Methods

4.1 |. Strains, Plasmids, Media

The bacterial strains and plasmids used in this study are listed in Table S1. S. flexneri strains were grown routinely at 30°C or 37°C with aeration (constant shaking at 325 rpm) in trypticase soy broth (TSB) or on Congo Red agar at 37°C (TSB containing 1.5% (w/v) agar and 0.01% (w/v) Congo Red (Sigma Chemical Co., St. Louis, MO)). The maintenance of the large virulence plasmid by S. flexneri was assessed by the ability to bind Congo Red on Congo Red agar. When required, antibiotics were added to growth media at the following final concentrations: ampicillin, 100 μg/mL; and chloramphenicol, 25 μg/mL. Growth conditions for specific reporter assays are provided in the “Reporter Assays” section.

4.2 |. Construction of Reporter Plasmids

All oligonucleotides and G-blocks (Integrated DNA Technologies) used in this study are listed in Table S2.

4.2.1 |. Construction of Mutated PicsP

To construct a PicsP-lacZ reporter that lacked the native VirB binding site (pMK48), a BglII and PstI fragment carrying the lambda oop terminator from pAFW04 was inserted into pMIC18 (Castellanos et al. 2009).

4.2.2 |. Construction of PicsP+1 kb

To construct PicsP-lacZ reporter plasmid derivatives, we began with pAFW04 (Basta et al. 2013; Table S1). To construct PicsP+1 kb (pJSR01), 1000 base pairs of the icsA coding region were amplified from S. flexneri 2a strain 2457T by polymerase chain reaction (PCR) using oligonucleotides W808 and W809. The intergenic region of the icsA gene was chosen to match the AT content of the icsP genetic locus into which it was inserted, without introducing regulatory elements. The resulting amplicon was cut with BglII and BamHI, and inserted in the reverse orientation into the unique BglII restriction site between the VirB binding site and the region required for H-NS-mediated silencing of the icsP promoter of pAFW04. In the resulting construct, the VirB binding site was pushed 1 kb upstream, centered 2137 bp upstream of the primary TSS of the icsP promoter.

4.2.3 |. Construction of PicsP-lacZ Distal Series

To create the PicsP-lacZ reporter with the wild-type VirB binding site repositioned to the opposite side of the plasmid (centered at −4759, relative to the PicsP TSS), the “distal site,” a three-step process was used. First, the wild-type VirB binding site from pAFW04 was amplified by PCR using W898 and W899; the amplicon was cut with XmnI and inserted into a pBlueScript KS II+ holding vector (pCC20) and confirmed by Sanger sequencing. Second, the XmnI insert, bearing 225 bp of the PicsP promoter, which included the VirB binding site, was inserted into pMK29 at the unique XmnI restriction site between the origin of replication and the cat gene to generate pCC18. Third, the icsP promoter with a mutated VirB binding site from pMK48 was then inserted into pCC18 using the XbaI and PstI restriction sites to produce pCC27. Consequently, pCC27 possesses a mutated site at the native position (−1137 bp) and a wild-type VirB binding site at the distal position (−4759), described as the distal VirB binding site construct in this work.

Two control plasmids for the distal VirB binding site construct were generated using a similar scheme. First, a 225 bp fragment of the PicsP promoter carrying the mutated VirB binding site from pMK48 was amplified by PCR using W898 and W899; the amplicon was cut with XmnI and inserted into a pBlueScript KS II+ holding vector (pCC19) and confirmed by Sanger sequencing. The resulting XmnI fragment, bearing 225 bp of the PicsP promoter, which included the mutated VirB binding site, was inserted into pMK29 to produce pCC17. Then, an icsP promoter sequence carrying either a wild type or mutated VirB binding site at its native position was introduced into pCC17, generating pCC23 and pCC25, respectively. Consequently, pCC23 possesses the wild-type VirB binding site at the native position (−1137) and a mutated VirB binding site at the distal position (−4759), while pCC25 possesses a mutated VirB binding site at both positions.

4.2.4 |. Construction of PicsP-lacZ Medial Series

To construct the PicsP-lacZ reporter with the VirB binding site repositioned to the “medial” position between the native (−1137) and distal (−4759) positions, the region between the cat gene and the lambda oop transcriptional terminator of pMK48 was targeted. G-Block 22 duplex DNA containing the 225 bp region used to make the distal wild-type site (in pCC27) and the DNA sequence located downstream of the BspEI site found within the CmR gene was inserted into a pBlueScript KS II+ holding vector (pCC42). After verifying by Sanger sequencing, the region of interest was cloned into pMK48 using BspEI to produce pCC44, a reporter with the wild-type VirB binding site centered 3317 bp upstream of the TSS of the icsP promoter and the mutated VirB binding site at the native position within the promoter region (−1137).

Two control plasmids for the medial VirB binding site construct were generated, starting with pCC42 (the holding vector containing G-Block 22; described previously). The mutated VirB binding site from pMK48 was amplified by PCR using oligonucleotides W936 and W937 and inserted into pCC42 to replace the wild-type site using XmaI and KpnI (pCC43). After verifying by Sanger sequencing, the now-modified G-Block sequence carrying the mutated VirB binding site was excised using BspEI and cloned separately into pAFW04 and pMK48 to produce pCC46 and pCC45, respectively. Consequently, pCC46 possesses the wild-type VirB binding site at the native position (−1137), and the mutated VirB binding site at the medial position (−3317) while pCC45 possesses only mutated VirB binding sites at both positions.

4.2.5 |. Construction of Distal Roadblocking Series

Construction of plasmids for the roadblocking experiments began with two duplex DNA G-Blocks, and a three-step process was used. G-Block 20 and 21 were designed to contain a centrally located VirB binding site with flanking sequence from the icsP promoter (5′-ATTTCAGTATGAAAT-3′) (330 bp in total) and tandem lacO sites centered either 100 bp upstream or downstream of the center of the VirB binding site, respectively. After inserting each duplex DNA into separate pBlueScript KS II+ holding vectors (pCC32 and pCC35, respectively) using XmnI and verifying their sequences, the respective regions of interest were excised using XmnI and separately inserted into the unique XmnI restriction site of pMK29 to produce pCC33 (tandem lacO sites upstream of the VirB binding site) and pCC36 (tandem lacO sites downstream of the VirB binding site). Finally, the icsP promoter carrying the mutated VirB binding site from pMK48 was inserted into pCC33 and pCC36 using XbaI and PstI to produce pCC34 and pCC37, respectively. Consequently, pCC34 and pCC37 possess lacZ under the control of the mutated icsP promoter region with a VirB binding site centered 4759 bp upstream and tandem lacO sites centered either 4858 bp (pCC34) or 4660 bp (pCC37) upstream, all relative to the primary TSS of PicsP.

4.2.6 |. Construction of In Vivo Remote Binding Tool and Derivatives

To make the remote binding tool and its derivatives, the VirB binding site from pAFW04 (Castellanos et al. 2009) was amplified by PCR using W598 and W89. The resulting amplicon was digested with BamHI and BglII and ligated into pBT-empty (pJAI16; Karney et al. 2019) digested with BglII. The resulting pRBT-WT (pADK06) was confirmed by BamHI and EcoRI digest to determine the orientation of the VirB binding site and further verified by Sanger sequencing (Genomics Core Facility; University of Nevada Las Vegas). pRBT+1 kb-WT (pJSR06) was made by PCR amplifying a fragment of the icsA gene from PicsP+1 kb (pJSR01) using W808 and W809. This amplicon was digested with BamHI and BglII and ligated into pRBT-WT digested with BglII. The orientation of the icsA fragment was verified by BglII and BamHI digestion, and the sequence was verified by Sanger sequencing. Both pRBT-mut and pRBT+1 kb-mut were made by digesting pMIC18 (Castellanos et al. 2009) with SalI and BglII and ligating the fragment carrying the mutated VirB binding site into pRBT-WT and pRBT+1 kb-WT, respectively.

4.3 |. Reporter Assays

4.3.1 |. lacZ Transcriptional Reporters and β-Galactosidase Assays

To measure VirB-mediated anti-silencing of the PicsP promoter, a variety of PicsP-lacZ transcriptional reporter plasmids were used (as described in the Results). In each of these reporters, the icsP promoter and the first 48 bp of the icsP coding region were cloned immediately upstream of three translation stop sites (one in each reading frame to ensure the promoter fusion was purely transcriptional) and a promoterless lacZ gene. Thus, lacZ expression in these constructs is directly regulated by the icsP promoter. To measure VirB-mediated interference of PT5, pRBT and pRB+1 kb and their respective derivatives were used. In each case, the PT5 promoter and 61 bp of the 5′ UTR were cloned immediately upstream of three translation stop sites and a promoterless lacZ gene, so in these constructs, lacZ expression is directly regulated by the T5-lac promoter. Promoter activities were determined by measuring β-galactosidase activity using a previously described protocol (Wing et al. 2004, adapted from Miller 1972); experiments were performed using biological triplicates (n = 3) in three independent trials, and a representative trial is given for each experiment.

4.3.2 |. Growth Conditions for Experiments Evaluating Regulation With Endogenous Levels of VirB

Reporter plasmids were independently introduced into either the wild-type S. flexneri (2457T) or the isogenic virB mutant (AWY3) by electroporation. Cultures were grown overnight (16 h) at 30°C in TSB, then diluted 1:100 and grown for 5 h at 37°C with aeration in TSB prior to cell lysis.

4.3.3 |. Growth Conditions for VirB Titration Assays

The virB mutant strain AWY3 carrying either the l-arabinose-inducible virB expression vector pATM324 (pBAD18-virB) or the empty vector control (pBAD18) was transformed with the reporter plasmids of interest. Cultures were grown overnight at 30°C in LB containing 0.2% d-glucose, diluted 1:100 in LB, and grown for 3 h at 37°C with aeration and then induced with l-arabinose at varying final concentrations (0.005%, 0.01%, 0.02%, 0.2%) or not induced (0%) and grown an additional 2 h prior to cell lysis.

4.3.4 |. Growth Conditions for Roadblocking Experiments

A previously used protocol was used (Weatherspoon-Griffin et al. 2018) to evaluate the effect of lac repressor binding on VirB-dependent regulation of the icsP promoter from the “distal” VirB binding site (−4759 from the TSS). 2457 T and the isogenic virB mutant, AWY3, carrying a lacIq expression vector (pQE2) and a reporter plasmid (pCC27, pCC37, pCC34, or pMK29), were induced for 3 h with 250 μM isopropyl β-d-1 thiogalactopyrano-side (IPTG) or not induced, and promoter activities were then measured.

4.3.5 |. Growth Conditions for In Vivo Remote Binding Tool Assays

pBT, pRBT, and their derivatives were introduced into the virB mutant strain AWY3 carrying the l-arabinose-inducible pBAD-virB (pHJW16) or pBAD-empty control (pHJW14) by electroporation and assayed as described previously (Karney et al. 2019). l-Arabinose concentration is 0.2% unless otherwise specified.

4.4 |. Quantification of Cellular VirB Levels by Western Blotting

To match the conditions used in reporter assays where endogenous levels of VirB were used, overnight cultures of 2457T were diluted 1:100 and grown in TSB for 5 h. Similarly, to set up conditions for subsequent titration experiments, overnight cultures of the virB mutant AWY3 carrying a PicsP-lacZ reporter and either pBAD18-virB or empty pBAD18 were diluted 1:100 and grown in LB for 3 h and then induced with varying concentrations of l-arabinose for 2 h. All cultures were normalized to cell density (optical density at 600 nm), washed with 0.2 M Tris buffer (pH 8.0), and resuspended in 200 μL 10 mM Tris (pH 7.4) with 1 mM phenyl-methylsulfonyl fluoride (PMSF). 50 μL of 5× SDS-PAGE loading buffer (containing β-mercaptoethanol) was added to each sample prior to boiling for 5–10 min. Equal volumes of normalized samples were separated by SDS-PAGE. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, and VirB was detected using an affinity-purified rabbit anti-VirB polyclonal antibody (obtained from Pacific Immunology) and a donkey anti-rabbit IgG-horseradish peroxidase (HRP; GE, NA9340) secondary antibody. Blots were imaged using the autoexposure setting with chemiluminescence detection on an Azure C400 imager (Azure Biosystems). Western blots were repeated twice using independent biological replicates. Densitometry of VirB levels was performed using AzureSpot analysis software.

4.5 |. Statistical Analysis

All statistical analyses were performed using IBM SPSS Statistics for Windows, version 28.01.0. Data were evaluated using one-way, two-way, or three-way Analysis of Variance (ANOVA) followed by Šidák or Bonferroni correction as indicated in figure legends, along with p values. Tables documenting all statistical comparisons (Tables S3–S9) are provided in Supporting Information.

Supplementary Material

Supp Mats for Cris et al., 2025

Additional supporting information can be found online in the Supporting Information section.

Acknowledgments

We thank lab members past and present for insightful discussions. This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIH) (R15 AI090573). UNLV Genomics Core Facility, used throughout this study, was supported by the INBRE Program of the National Center for Research Resources (P20 RR-016464). ENH was supported by an NIH diversity supplement (R15-AI090573-05S1). The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views of NIH. These funding sources had no role in the study design, data collection, and interpretation, or the decision to submit the work for publication.

Funding:

This work was supported by the National Center for Research Resources (Grant P20 RR-016464) and the National Institute of Allergy and Infectious Diseases (Grants R15 AI090573 and R15 AI090573-05S1).

Footnotes

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

A local data repository called the “X drive” belonging to the College of Sciences at the University of Nevada, Las Vegas, will be used to archive data related to this project. Data will be copied into “publication” folders 4 months prior to publication and stored in perpetuity. Relevant data for this publication are included in Supporting Information.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Mats for Cris et al., 2025
NIHMS2124066-supplement-Supp_Mats_for_Cris_et_al___2025.pdf (440.4KB, pdf)

Data Availability Statement

A local data repository called the “X drive” belonging to the College of Sciences at the University of Nevada, Las Vegas, will be used to archive data related to this project. Data will be copied into “publication” folders 4 months prior to publication and stored in perpetuity. Relevant data for this publication are included in Supporting Information.

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