The small protein RmpD drives hypermucoviscosity in Klebsiella pneumoniae
aDepartments of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC 27599
bGenetics, University of North Carolina School of Medicine, Chapel Hill, NC 27599
1Corresponding author Email: kawalker@med.unc.eduABSTRACT
Klebsiella pneumoniae has a remarkable ability to cause a wide range of human diseases. It is divided into two broad classes: Classical strains that are a notable problem in healthcare settings due to multidrug resistance, and hypervirulent (hv) strains that are drug sensitive, but able to establish disease in immunocompetent hosts. Alarmingly, there has been an increased frequency of clinical isolates that have both drug resistance and hv-associated genes. One such gene is rmpA that encodes a transcriptional regulator required for maximal capsule (cps) gene expression and confers hypermucoviscosity (HMV). This link has resulted in the assumption that HMV is caused by elevated capsule production. However, we recently reported a new cps regulator, RmpC, and ΔrmpC mutants have reduced cps expression but remain HMV, suggesting that capsule and HMV may be separable traits. Here, we report the identification of a small protein, RmpD, that is essential for HMV, but does not impact capsule. RmpD is 58 residues with a putative N-terminal transmembrane domain and highly positively charged C-terminal half, and it is conserved among other hv K. pneumoniae strains. Expression of rmpD in trans complements both ΔrmpD and ΔrmpA mutants for HMV, suggesting that RmpD is the key driver of this phenotype. The rmpD gene is located between rmpA and rmpC, within an operon regulated by RmpA. This data, combined with our previous work, suggests a model in which the RmpA-associated phenotypes are largely due to RmpA activating the expression of rmpD to produce HMV and rmpC to stimulate cps expression.
SIGNIFICANCE
Capsule is a critical virulence factor in Klebsiella pneumoniae, in both antibiotic-resistant classical strains and hypervirulent strains. Hypervirulent strains usually have a hypermucoviscous (HMV) phenotype that contributes to their heightened virulence capacity, but the production of HMV is not understood. The transcriptional regulator RmpA is required for HMV and also activates capsule gene expression, leading to the assumption that HMV is caused by hyperproduction of capsule. We have identified a new gene (rmpD) required for HMV but does not contribute to capsule production. This distinction between HMV and capsule production will promote a better understanding of the mechanisms of hypervirulence, which is in great need given the alarming increase in clinical isolates with both drug resistance and hypervirulence traits.
INTRODUCTION
Klebsiella pneumoniae has classically been considered an opportunistic pathogen associated with infection of immunocompromised patients in nosocomial settings (1, 2). Most infections are caused by classical K. pneumoniae (cKp) strains and present as pneumonias or urinary tract infections, sometimes leading to bacteremia and septic shock. The widespread occurrence of extended-spectrum β-lactam resistant and carbapenem resistant strains has led both the CDC and WHO to categorize K. pneumoniae at the highest level of concern for antibiotic resistant threats (3, 4). In addition, colistin and tigecycline resistant strains of K. pneumoniae have been isolated, severely limiting treatment options (5). The deadly case of a pan-resistant cKp strain (resistant to 26 antibiotics) underscores the immense challenge of treating Klebsiella infections (6).
In contrast to cKp, the hypervirulent K. pneumoniae (hvKp) are community-acquired by immunocompetent individuals (7). HvKp pathology is more severe than that typical of cKp and can include pyogenic liver abscesses, necrotizing fasciitis, meningitis, and endophthalmitis (8, 9). Of particular concern is the emergence of strains with both hypervirulent (hv)-associated genes or traits and the multi-drug resistance that is characteristic of cKp (10). Antibiotic resistance genes are often encoded on plasmids (11, 12) and many of the genes corresponding to hypervirulence are carried on large virulence plasmids or mobile genetic elements incorporated on the chromosome (13-15). That these genetic entities can be horizontally transferred suggests there is an increased risk of strains acquiring both hypervirulence and multidrug resistance (16, 17). Alarmingly, there have been recent reports of extensively resistant hypervirulent K. pneumoniae (18, 19), and multiple strains where both hv-associated genes and antimicrobial resistance genes were present on the same mobile vector have been documented (20-23). These reports of convergence of hypervirulence and antimicrobial resistance in the same strain have heightened the need to better understand how hypervirulence genes interface with a strain’ s genetic background to confer hypervirulent phenotypes. This is particularly important given the extensive genetic diversity of genetic content between K. pneumoniae strains.
Klebsiella virulence is largely attributable to LPS, pili, a polysaccharide capsule, and siderophores, and these are present in virtually all pathogenic strains (2), heavy metal resistance (24) and hypermucoviscosity (HMV) (2, 25, 26). Capsule is also linked to hypervirulence as the majority of hvKp have type K1 or K2 (27), although hv-associated traits have been found in strains with other capsule types (28). Compared to cKp, hvKp produce a thick ‘hypercapsule’ that is thought to contribute to the HMV phenotype.
RmpA is a LuxR-like transcriptional regulator frequently encoded on virulence plasmids or on integrative chromosomal elements (ICEKp) and was initially discovered as a regulator of HMV (14, 25). While the strong correlation between the presence of rmpA and hypervirulence has made it a key biomarker for hvKp (27, 29), we still know very little about how rmpA contributes to HMV and hypervirulence. Previous studies established that loss of rmpA decreases capsule (cps) gene expression and reduces HMV in commonly used hvKp strains (30, 31). We recently confirmed these rmpA-dependent phenotypes in strain KPPR1S (26). We also described another regulator of capsule gene expression, RmpC, which is encoded downstream of rmpA; rmpA and rmpC are cotranscribed from the same promoter that is positively regulated by RmpA (26). Like rmpA mutants, the rmpC mutant showed reduced cps gene expression but, unlike rmpA mutants, retained HMV. We further showed that overexpression of rmpA in WT or the ΔrmpA and ΔrmpC mutants increased HMV. However, overexpression of rmpA did not restore cps expression in the ΔrmpC strain, and overexpression of rmpC elevated cps expression even in the ΔrmpA strain (26). These data suggest that: 1) RmpA is an important determinant for HMV but RmpC is not, 2) reduced cps expression in the ΔrmpA strain is likely a consequence of reduced rmpC expression rather than direct regulation by RmpA, and 3) high levels of cps expression are not necessary to confer HMV. This latter conclusion stems from the fact that the ΔrmpC mutant has reduced cps expression but retains the HMV phenotype, and that exogenous expression of rmpA in the ΔrmpC strain results in elevated HMV without restoring cps expression. Importantly, this was the first clear evidence of a separation between capsule expression and HMV and suggests that HMV is not simply a consequence of elevated capsule production.
Furthering this work, we report here the discovery of a small protein, RmpD, encoded between rmpA and rmpC that is essential for HMV. The ΔrmpD mutant is non-HMV, has no change in cps expression, and produces the same amount of uronic acid (capsule) as the wild type parental strain. This provides additional evidence that HMV and capsule result from distinct processes. Expression of rmpD is sufficient to confer HMV to a ΔrmpA mutant. It is transcribed by the promoter upstream of rmpA and therefore is also regulated by RmpA. Thus, it appears that the loss of HMV and cps expression observed in rmpA mutants is due to reduced transcription of rmpD and rmpC, respectively, and that the contribution of RmpA to these phenotypes is as an activator of this operon.
RESULTS
RmpD is required for hypermucoviscosity
Having made the observation that hypermucoviscosity (HMV) is not necessarily a consequence of elevated capsule expression from examining the individual ΔrmpA and ΔrmpC strains with complementation plasmids (26), we took one further step by similarly testing a strain that lacked the region encoding both rmpA and rmpC (strain ΔrmpA-C). We predicted that introduction of pRmpA would restore HMV and that pRmpC would restore manC expression. HMV was assessed by measuring the OD600 of culture supernatants following low-speed centrifugation and expression of manC was monitored using a promoter-GFP reporter (manC encodes an enzyme that makes one of the K2 sugar precursors and is located in the cps locus). While pRmpC resulted in elevated manC levels as expected, pRmpA failed to restore HMV (Fig. 1A, B). However, introduction of a plasmid containing the entire region that was deleted resulted in elevated HMV, and in normal levels of manC expression (as previously observed), suggesting an element contained within the intergenic space was necessary for HMV. In examining this region, we noted a predicted ORF in the 375 bp region between the rmpA and rmpC genes (VK055_5098), but this predicted ORF is encoded on the opposite strand (Fig. 1C). The rmp locus encoded on the virulence plasmid of NTUH-K2044 also indicates a predicted ORF downstream of rmpA (KP1_p021), but this one is encoded on the same strand. The DNA sequence of these loci is very similar between KPPR1S and NTUH-K2044, and the ORF prediction analysis in Geneious R11 identified an ORF in KPPR1S nearly identical to KP1_p021. Thus, we cloned both predicted ORFs from KPPR1S into pMWO-078, transformed them into KPPR1S, ΔrmpA, ΔrmpC and ΔrmpAC strains, and assayed for HMV (Fig. 1D). Introduction of pORF_5098 did not alter HMV, but the KPPR1S homolog of KP1_p021 (pRmpD) resulted in a hyper-HMV phenotype in all strains, including the ΔrmpAC strain. Thus, this gene is required for HMV and was named rmpD. The region containing rmpD is within the rmp operon (Fig. S1). Although there is a predicted ORF of 58 amino acids in the DNA sequence cloned in pRmpD, there remained the possibility that this region encoded a regulatory RNA. To distinguish between these possibilities, we generated a plasmid with a rmpD-2xFLAG fusion and were able to detect a FLAG-tagged protein of the predicted size (Fig. 1E), indicating that rmpD encodes a protein and not a regulatory RNA.
To further analyze the role of RmpD, we constructed a strain lacking rmpD (ΔrmpD) and examined manC expression and HMV in this mutant. The ΔrmpD mutant had wild-type levels of manC expression and was non-HMV (Fig. 2). Supporting the notion that it is rmpD and not rmpA that is necessary for HMV, pRmpA was unable to restore HMV in the ΔrmpD mutant. Introduction of pRmpC into the ΔrmpD strain resulted in the same high levels of manC expression observed in other strains but did not restore HMV. Complementation of ΔrmpD with pRmpADC (formerly pRmpA-C) also resulted in an elevated level of HMV. The cultures in which rmpD is overexpressed become extremely viscous and have the consistency of a thick syrup (Fig. S2), but have no change in transcription of manC (Fig. S3). Collectively, these data suggest that RmpD, rather than RmpA, is necessary for HMV. Given that RmpA regulates the promoter driving expression of rmpADC (26), the well-established role of RmpA as a requisite factor for the HMV phenotype is likely due to its function as a transcriptional activator of rmpD expression.
Impact of rmpD in capsule mutants
Contributing to assumptions in the field that HMV is derived from capsule, mutants in hvKp strains that produce no or reduced levels of capsule have also typically been non-HMV (30-33). To further probe the distinction between capsule and HMV, we transformed two capsule mutants (ΔmanC, ΔwcaJ) with pRmpD to determine if these strains could become hyper-HMV. manC encodes a GDP-mannose pyrophosphorylase that produces UDP-mannose, one of the sugar precursors of K2 capsule, and wcaJ encodes the initiating glycosyltransferase (undecaprenyl phosphotransferase) involved in building the four-sugar K2 subunit. Both capsule mutants, with or without pRmpD, fully sedimented following low speed centrifugation (Fig. 3A), suggesting that the HMV phenotype requires some capsule biosynthetic enzymes and may require capsule production. We therefore examined capsule production in the ΔrmpD strain using the uronic acid (UA) assay. Curiously, there was no decrease in UA levels in the ΔrmpD strain compared to WT, and addition of pRmpD did not lead to increased UA (Fig. 3B). Collectively, these data imply that production of capsule is not impacted by RmpD, but that at least some components of capsule must be present in order to become HMV. The negative stain, India ink, was used to visualize capsule. WT bacteria show exclusion zones that vary somewhat in size, whereas the rmpD-deficient bacteria have thinner, uniform clearing zones (Fig. 4). When rmpD is overexpressed in either strain, the bacteria have uniformly large exclusion zones. As predicted, no exclusion zones were observed from staining of ΔmanC bacteria, although the field has ample bacteria present (Fig. S4). Given that there is no difference in the amount of UA between the WT and ΔrmpD strains, these data suggest that the material forming the abundant exclusion zones is different than a typical UA-containing capsule.
There are several known regulators of capsule gene expression, of which our lab has identified three and studied five (26, 32). These mutants (ΔrmpA, ΔrmpC, ΔkvrA, ΔkvrB, and ΔrcsB) all have reduced UA levels and capsule expression, and all but ΔrmpC are non-HMV. To further probe the factors necessary for HMV, we transformed the ΔkvrA, ΔkvrB, and ΔrcsB strains with pRmpD and assessed HMV and capsule phenotypes. Each mutant had WT-like HMV, elevated UA levels and elevated manC expression with the respective deleted gene complemented in trans (Fig. S5). With pRmpD, the ΔkvrB and ΔrcsB strains became hyper-HMV similarly to the WT strain, and an intermediate level of HMV was observed for the ΔkvrA strain (Fig. 3C). Consistent with the results presented above, pRmpD did not restore UA production in these mutants (Fig. 3D), further implying that strains with low capsule expression and UA production are still capable of becoming HMV.
rmpD contributes to immune evasion
One of the virulence phenotypes associated with capsule is the blocking of adherence and phagocytosis (34). To determine if HMV specifically contributed to these processes, we performed adherence assays with the macrophage-like J774A.1 cells. Bacterial strains with pRmpD or vector were grown in the presence of inducer to express rmpD, then allowed to interact with J774A.1 cells for 30 minutes. The cells had been pre-treated with cytochalasin D to prevent phagocytosis, allowing measurement of attachment only. After rinsing, the cells were lysed, and each sample was diluted and plated for bacterial enumeration. The WT strain showed about 5% adherence (normalized to inoculum), and the ΔmanC mutant showed nearly 70% adherence (Fig. 5). The ΔrmpD strain behaved like the acapsular manC mutant, with ∼85% adherence. The WT or ΔrmpD strains with pRmpD were virtually non-adherent, with less than 1% of the bacteria attached. This reduction was not observed in the ΔmanC mutant with pRmpD, most likely because it remains non-HMV. Because the ΔrmpD strain still produces capsule at the level of WT, and the attachment phenotype is the same as a capsule mutant, it appears that the HMV phenotype is the main factor blocking attachment to host cells, and not capsule. Whether capsule has any protective role against adherence, and likely phagocytosis, cannot be fully ascertained from these results, but it is clear that HMV is important as the hyper-HMV strains were almost completely non-adherent.
Sequence and functional conservation of RmpD homologs
Examination of other hvKp strains reveals that rmpD is present when rmpA and rmpC are present. There are three other hvKp strains that are frequently used for the study of capsule expression and the RmpD proteins in these strains share a high degree of identity (Fig. 6A). To test for functionality, the rmpD gene from each of these strains was cloned and expressed in WT and ΔrmpD strains of KPPR1S. Not surprisingly, each gene retained the ability to confer hyper-HMV in both WT and ΔrmpD strains (Fig. 6B), suggesting that the role of RmpD in HMV is conserved among varying K. pneumoniae isolates.
DISCUSSION
Hypermucoviscosity (HMV) is a phenotype possessed by a subset of K. pneumoniae strains and is one of the phenotypes associated with hypervirulent strains (2). RmpA has been established as an essential factor for HMV, and rmpA mutants also show reduced capsule gene expression (26, 30, 31). Thus, it has long been assumed that the HMV phenotype was a consequence of abundant capsule production in excess of that observed in classical strains. This arose despite statements in early studies that HMV did not appear to be linked to capsule production (25, 35). However, FITC staining of an hv K. pneumoniae strain incubated with K2 antisera suggested the extracapsular substance associated with HMV contained capsular material (36). Although it has been 30 years since the discovery of RmpA, no direct regulation by RmpA of cps expression (or other genes) has been demonstrated. In our investigations into the contributions of RmpA to hypervirulence, we confirmed its role in HMV and cps expression, but also ascertained that the mechanisms contributing to these phenotypes is much more complex than had been presumed (26). We identified a downstream gene encoding RmpC, a putative transcriptional regulator that modulates cps expression, and found that rmpA and rmpC are in an operon that is autoregulated by RmpA. RmpA and RmpC have overlapping and distinct functions, most notably that the ΔrmpA mutant is non-HMV but the ΔrmpC mutant retains HMV. Both mutants have a similar reduction in cps expression, however, overexpression of rmpC complements cps expression even in strains lacking rmpA. While RmpC has also not been demonstrated to directly regulate cps promoters, this data indicated that RmpA was not likely to be a direct regulator of the cps genes. We thus concluded that RmpA controlled HMV while RmpC controlled cps expression in work that provided the first clear evidence separating the phenotypes of HMV and capsule levels.
In evaluating cps expression and HMV in what we thought was a double ΔrmpA-rmpC mutant it became clear that the story was not as simple as suggested by the analysis of individual rmpA and rmpC mutants. Namely, pRmpA did not restore HMV to this ΔrmpA-C mutant but a plasmid containing the entire deleted region (pRmpADC) did restore HMV (pRmpC does restore cps expression in this mutant). In this current study, we report the initial characterization of RmpD, a small protein encoded in the region between rmpA and rmpC, also within the rmp operon. The data presented here suggest that RmpD is the key factor driving the HMV phenotype. Collectively, our data supports a model in which the role played by RmpA in the HMV and cps expression phenotypes is to activate expression of rmpD and rmpC. This is evidenced by 1) the restoration of HMV in the ΔrmpA and ΔrmpADC strains with pRmpD, and restoration of cps expression in the ΔrmpA and ΔrmpADC strains with pRmpC, and 2) the inability of pRmpA to restore HMV in the ΔrmpD strain or cps expression in the ΔrmpC strain. Given that several RmpD orthologs were able to complement HMV in the ΔrmpD strain, and that rmpD is present in strains that also have rmpA and rmpC, we speculate that RmpD is part of a conserved mechanism conferring HMV to K. pneumoniae.
Several lines of evidence further support the notion that production of capsule and HMV are separable. First, deletion of rmpD did not alter UA levels, suggesting that production of the capsular material is unaffected by this mutation. Second, strains that are hyper-HMV from overproduction of RmpD did not produce more UA than the WT strain. Third, trans expression of rmpD in the regulatory mutants (ΔrmpA, ΔkvrA, ΔkvrB, and ΔrcsB), that all have reduced cps expression and capsule production, were all complemented for HMV. Each of these regulators activate transcription of the rmpADC promoter; thus, the loss of HMV in these mutants is most likely due to reduced expression of rmpD. Curiously, even though we can detect almost no expression from the manC promoter in the ΔrcsB strain (26), introduction of pRmpD in the ΔrcsB mutant, but not in the ΔmanC mutant, results in hyper-HMV. Either very low levels of mannose-1-phosphate guanylyltransferase are sufficient for HMV production, or HMV does not actually require this enzyme and the HMV defect in a ΔmanC strain is an indirect effect of loss of this gene.
In mucosviscosity and adherence assays, the ΔrmpD strain behaves nearly identically to the capsule mutant ΔmanC. Both mutants pellet tightly and are highly adherent to host cells. The hyper-HMV strains (WT and ΔrmpD with pRmpD) are essentially non-adherent, but the non-HMV ΔmanC + pRmpD strain remains highly adherent. This raises the question as to whether or not the anti-adherence property is dependent on capsule or on HMV. Given that the ΔrmpD strain is encapsulated, it appears that HMV is a more critical determinant for blocking adherence, and quite likely, in blocking phagocytosis as well. This is consistent with the non-HMV ΔrmpA strain having a more severe virulence defect than the HMV-positive ΔrmpC strain in the mouse pneumonia model (26). The limited data on the adherence and anti-phagocytic properties of cKp strains makes it difficult to fully extrapolate the significance of capsule in these processes. As HMV has been established as contributing to virulence, rmpD mutants are likely to be attenuated in vivo. Support for this comes from re-examination of the virulence defects of the ΔrmpA and ΔrmpC strains. While it is possible that RmpA regulates additional virulence factors, the loss of rmpD expression in the ΔrmpA mutant likely contributes to the stronger virulence defect in the ΔrmpA mutant than that observed from the ΔrmpC mutant. Similarly, analysis of KPPR1 genes essential for infection in a mouse pneumonia model identified mutations in VK055_5096 as deficient for virulence (37). This orf is located immediately upstream of rmpA (VK055_5097) and the transposon insertion quite likely impaired expression of the rmp locus. Furthermore, the virulence plasmid-encoded rmpD gene (along with rmpA and rmpC) was found to be associated with liver abscess formation by NTUH-K2044 (38).
Complicating the notion that HMV is not simply a consequence of overabundant capsule production is that hyper-HMV did not occur in capsule-deficient mutants carrying pRmpD. This suggests that strains can be capsule-positive/HMV-positive or capsule-positive/HMV-negative, but not capsule-negative/HMV-positive. One possible explanation for this is that the HMV material is capsular, but that its export is altered in the presence of RmpD. This situation would mean that even reduced levels of biosynthetic enzymes such as those found in the regulatory mutants are sufficient to yield the extra polysaccharides. A second explanation is that HMV is a polysaccharide distinct from capsule, but that some cps-encoded functions are required to produce this material. A third possibility is that the HMV material is a modified form of capsule. The presence of RmpD could influence synthesis or export of the altered polysaccharide. That capsule-like material is part of HMV material is supported by the K2-positive staining of the HMV substance from a WT strain but not from non-HMV mutants (36).
To date, HMV has primarily been associated with hv K1 and K2 strains, but more than 130 capsule types of K. pneumoniae have been identified (39). Of significant concern is the number of recent reports of strains with both carbapenem resistance and hv-associated genes, including rmpADC. These strains are genetically quite distinct (including capsule type) from the hvKp that have been circulating, and it is not known to what degree acquisition of the rmpADC locus will impact HMV and virulence of these strains. While we have shown that RmpD from either a K2 or K1 strain can confer HMV in a K2 strain, it is not clear if there is capsule type specificity for this RmpD function. We also do not know, beyond a few cps genes, what, if any, other genes are necessary to confer HMV or if these genes are conserved in all K. pneumoniae strains. A better understanding of what is required for HMV and how genetic background influences the HMV associated hypervirulent phenotypes will be important for determining the risks associated with CR-cKp strains that acquire rmpADC.
MATERIALS and METHODS
More detail can be found in the Supplementary Information
Bacterial strains, plasmids and growth conditions
The strains and plasmids used in this work are listed in Table S1. E. coli strains were grown in LB medium at 37°C. K. pneumoniae were grown at 37°C in M9 medium supplemented with 0.4% glucose and 0.2% casamino acids (M9-CAA). Unless otherwise noted, saturated overnight cultures were diluted to OD600 = 0.2 and grown for 6 h. Antibiotics were used where appropriate: kanamycin (Kan), 50 μg/ml; rifampicin (Rif), 30 μg/ml, spectinomycin (Sp), 50 μg/ml. For expression of genes cloned into pMWO-078, 100 ng/ml anhydrous tetracycline (aTc) was added to the media at the time of subculture. The primers used for cloning are listed in Table S2. In-frame gene deletions in K. pneumoniae were constructed by allelic exchange using pKAS46-based plasmids as described (26).
Complementation plasmids were constructed using pMWO-078 (40). Plasmids containing promoter-gfp fusions were cloned in pPROBE-tagless (41). The gfp reporter and complementation plasmids were introduced into K. pneumoniae by electroporation as described (26).
Transcriptional gfp reporter assays
Relative fluorescent units (RFU) and OD600 were measured from bacterial cultures diluted 1:10 using a Synergy H1 plate reader (Bio-Tek, Winooski, WI) and a Bio-Rad spectrophotometer (Bio-Rad, Hercules, CA), respectively. Data are presented as RFU/OD600, normalized to the activity from the wild type strain in each assay.
Assessment of capsule production and HMV
Immunoblotting
Whole cell lysates from cultures grown in M9-CAA with aTc for 6 h were separated on 15% SDS-PAGE gels, transferred to PVDF membranes and probed with α-FLAG antibody (Sigma, M2 monoclonal antibody) and detected with chemiluminescence.
Adherence assays
Adherence assays were performed essentially as described (32) using J774A.1 cells. The cells were pretreated with cytochalasin D 1 h prior to inoculation to prevent internalization of the bacteria. The adherent bacteria (recovered CFU) are reported as a percent of the inoculum CFU.
India ink staining
Bacterial cultures carrying a constitutively expressing gfp reporter (pJH026) were grown as for all other assays. Equal volumes of culture and India ink were mixed on a glass slide and overlaid with a coverslip. Microscopy was performed using a Keyence BZ-X810 microscope at 1000x magnification. Images vary some due to the irregular spreading of the liquid on the slide.
Statistics and Replicates
Statistical tests for each experiment are given in the figure legends and were performed using GraphPad Prism 8.2. In every assay, a minimum of three assays were performed, each with biological replicates. Typically, a representative experiment is presented.
ACKNOWLEDGEMENTS
We thank Rita Tamayo for thoughtful discussions on this manuscript and for use of the Keyence BZ-X810 microscope. This work was supported by R21AI132925 to V.L.M. from NIAID.