Plasmid-Chromosome Crosstalk in Staphylococcus aureus: A Horizontally Acquired Transcription Regulator Controls Polysaccharide Intercellular Adhesin-Mediated Biofilm Formation

Introduction

Staphylococcus aureus is a common human and animal pathogen, causing a wide range of clinical manifestations (Tong et al., 2015; Ballhausen et al., 2017). Due to the capability to readily acquire many different resistance genes, S. aureus and other staphylococcal species are regarded as pathogens of concern for public health (Foster, 2017; Lakhundi and Zhang, 2018). Thus, methicillin-resistant S. aureus (MRSA) and coagulase-negative staphylococci (MR-CoNS) are among the most common causes of healthcare-associated infections (Lee et al., 2018a; Becker et al., 2020). In this respect, the ability to form biofilms on the inert surfaces of medical devices is considered as important pathomechanism that contributed to the establishment of staphylococci as notorious nosocomial pathogens (Heilmann et al., 2019; Becker et al., 2020; Schilcher and Horswill, 2020). Biofilms are understood as bacterial communities that adhere to surfaces by encasing into a self-produced extracellular polymeric matrix (Costerton et al., 1999). The staphylococcal biofilm matrix may contain exopolysaccharides (Heilmann et al., 1996) and proteins (Rohde et al., 2005) as well as extracellular (e)DNA (Qin et al., 2007) [for a recent review see reference (Schilcher and Horswill, 2020)]. The key exopolysaccharide component of staphylococcal biofilms is PIA (polysaccharide intercellular adhesin), a beta-1,6 linked N-acetyl glucosaminoglycan, whose synthesis enzymes are encoded by the ica (intercellular adhesin) locus [recently reviewed in (Nguyen et al., 2020)]. PIA/ica was originally discovered in Staphylococcus epidermidis and was later also detected in S. aureus and other staphylococcal species (Heilmann et al., 1996; Mack et al., 1996; Cramton et al., 1999). Interestingly, ica locus homologs also exist in phylogenetically unrelated bacteria such as Escherichia coli (Wang et al., 2004), suggesting an eminent role of the factor in the evolution of bacterial biofilm functions. In these organisms, PIA is also often referred to as PNAG (poly-1,6-N-acetylglucosamine). The staphylococcal ica locus consists of two divergently oriented transcription units, one comprising the icaADBC operon (encoding the enzymes required for PIA synthesis) and the other harboring icaR which codes for a transcription factor of the TetR family (Figure 1A). IcaR, for which the crystal structure was solved, binds to a region upstream of icaA and represents a potent repressor of icaADBC operon transcription (Conlon et al., 2002a; Jefferson et al., 2004; Jeng et al., 2008). Regulation of the ica locus is highly complex and a plethora of environmental cues are known to influence PIA production many of which either directly or indirectly influencing icaR transcription (Conlon et al., 2002b; Cerca et al., 2008; Fey and Olson, 2010; Cue et al., 2012; Hoang et al., 2019; Nguyen et al., 2020). Expression of icaR is further controlled post-transcriptionally through RNA-mediated mechanisms that influence stability and translation of the icaR mRNA, with direct consequences for PIA production and biofilm formation (Ruiz de los Mozos et al., 2013; Rochat et al., 2018; Bronesky et al., 2019; Lerch et al., 2019; Schoenfelder et al., 2019). While nearly all S. aureus genomes carry the ica locus, distribution of the gene cluster among S. epidermidis and other CoNS species is more diverse and often associated with distinct clonal lineages (Kozitskaya et al., 2005; Conlan et al., 2012; Thomas et al., 2014; Méric et al., 2015; Méric et al., 2018; Lee et al., 2018b; Espadinha et al., 2019). The ica locus is usually located in the bacterial chromosomal DNA in all staphylococcal species. Previously, however, we detected an ica gene cluster of unknown genetic origin on plasmid pAFS11 in the bovine MRSA isolate S. aureus Rd11 (Feßler et al., 2017). S. aureus Rd11 is a livestock-associated (LA)-MRSA strain of sequence type ST398, a clonal lineage known for its potential to carry a broad range of both common and novel antibiotic resistance genes, many of which located on plasmids (Kadlec et al., 2012; Feßler et al., 2018). On pAFS11, antimicrobial and heavy metal resistance genes were found to be co-localized with a novel ica gene cluster. The ica locus on pAFS11 differed both on nucleotide and protein levels from the copy in the S. aureus Rd11 chromosome, and initial analyses (i.e. BLAST searches against the entire non-redundant sequence collections at NCBI) suggested that the plasmid-borne ica locus might have its origin in the CoNS species Staphylococcus sciuri [recently re-classified as Mammaliicoccus sciuri (Madhaiyan et al., 2020)] (Feßler et al., 2017). The mosaic structure of pAFS11 further suggests that the plasmid arose by a series of recombination events and was acquired by S. aureus Rd11 through horizontal gene transfer (HGT). As a result, S. aureus Rd11 carries two ica loci. Surprisingly, however, the strain did not produce biofilm when tested in standard tissue culture plate assays. Also, transformation of the pAFS11 plasmid into another S. aureus strain did not prompt biofilm formation, but even slightly reduced it (Feßler et al., 2017). In this study, we address the molecular mechanism underlying the biofilm-negative phenotype of pAFS11-bearing S. aureus. We identified an unexpected IcaR-mediated regulatory crosstalk between the plasmid-borne and chromosomally encoded ica loci, resulting in downregulation of biofilm formation. We discuss these findings in the context of co-evolution of virulence and resistance traits and raise the question of how genes newly acquired by HGT might become integrated into the regulatory network of host bacteria.

FIGURE 1

Figure 1 Conservation of pAFS11 ica locus. (A) TOP: schematic view of the organization of the ica locus including the IcaR tetramer and its function as repressor of transcription. BOTTOM: average distances between the ica loci of S. epidermidis O-47, S. epidermidis RP62A, S. aureus RN4220, S. aureus lineage ST398, S. sciuri NS1 and plasmid pAFS11. (B) Pairwise Alignment shown as percentage identity of nucleotide positions between the ica loci of pAFS11 vs. NS1, pAFS11 vs. RN4220 and RN4220 vs. RP62A. (C) Pairwise Alignment shown as percentage identity of amino acid residues between the Ica proteins of RN4220 vs. pAFS11 and RN4220 vs. RP62A. (D) Multiple sequence alignment of IcaR protein from pAFS11, RN4220, RP62A and O-47. Conservation is visualized as a histogram and a score is given for each column: conserved residues are indicated by ‘*’, and columns with residues, where all properties are conserved are marked with ‘+’. Putative icaA operon- interacting residues on IcaR in S. epidermidis are marked with a red triangle on top of the sequence (Jeng et al., 2008). All comparisons shown in (A–D) were calculated with the aid of Jalview (Waterhouse et al., 2009) from CLUSTAL Omega multiple sequence alignments (Madeira et al., 2019).

Materials and Methods

Sequence Alignments and Data Base Searches

Alignments of the nucleotide sequences of ica loci as well as that of amino acid sequences of Ica proteins from different species were performed with CLUSTAL Omega multiple sequence alignments (https://www.ebi.ac.uk/Tools/msa/clustalo) (Madeira et al., 2019). Average distance between the ica loci and pairwise alignments were calculated with the aid of Jalview (Waterhouse et al., 2009). Strains and sequences included into the analyses comprised S. epidermidis RP62A (accession no. NC_002976), S. epidermidis O-47 (accession no. CP040883), S. aureus ST398 (accession no. AM990992.1), S. aureus RN4220 (accession no. AFGU01000118.1), S. sciuri NS1 (accession no. LDTK01000031.1) and plasmid pAFS11 (accession no. FN806789.3). Data base queries with nucleotide and protein sequences were performed using the Basic Local Alignment Search Tool available at the National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Plasmid and Strain Construction

Strains, plasmids and oligonucleotides used for this work are listed in Tables 1 and 2, respectively.

TABLE 1

Table 1 Strains and plasmids.

TABLE 2

Table 2 Oligonucleotides.

Construction of a Markerless icaADBC Mutant

The markerless ica mutant was obtained via allelic replacement with inducible counter-selection using the pBASE6 shuttle vector (Bae and Schneewind, 2006; Geiger et al., 2012). pBASE6 vector was linearized using primers SLIC_pBASE_R and SLIC_pBASE_F. Total deletion was achieved by overlapping PCR using as template gDNA from RN4220 with primers Flank_A_SLIC together with Flank_A_rev and Flank_B_rev together with Flank_B_SLIC. The amplicon was introduced into the linearized pBASE6 vector using the in vivo E. coli cloning (iVEC) method (Nozaki and Niki, 2019). The resulting plasmid (pAK17) was transformed into the restriction-deficient strain RN4220. Mutagenesis was performed as described elsewhere (Bae and Schneewind, 2006). The deletion was verified by PCR with oligonucleotides spanning the deletion region.

Construction of Complementation Plasmids

All plasmids were created following the iVEC method (Nozaki and Niki, 2019). As iVEC turned out to be more efficient in the presence of buffer, the ligation buffer from the QuickLigation™ Kit (NEB, #M2200S) was added to the reactions. Sanger sequencing was used to verify accuracy of all plasmids. To create plasmids pGM10 and pGM12, the ica_pAF and ica_RN operons were amplified from pAFS11 and RN4220, respectively, and introduced in the linearized pRB473. Deletion of icaR coding regions from plasmids pGM10 and pGM12 (resulting in plasmids pGM11 and pGM13, respectively) was achieved by overlapping PCRs which amplified the respective vectors in two fragments that overlapped in the icaR deletion and multiple cloning site regions. Of note, this approach left putative icaR 5’ and 3’ untranslated regions (UTRs) intact which might be involved in post-transcriptional regulation of icaR. icaR_pAF was amplified from pAFS11 and introduced in the linearized pGM12, resulting in plasmid pGM14. icaR_RN was amplified from RN4220 and introduced in the linearized pGM10, resulting in plasmid pGM15. The vectors containing mutated sequences which alter the palindromes (pGM16 to pGM21) were generated by PCR site-directed mutagenesis amplifying the original vectors (see Table 2 for details) in two fragments that overlapped in the region containing the mutated palindrome and in the multiple cloning site region. In Table 2, the mutated palindrome sequences are shown as lower-case characters in the primer sequences.

Preparation of Total RNA and qRT-PCR

Total RNA of bacteria was isolated as described previously (Lerch et al., 2019). Briefly, RNA was precipitated with 1x volume isopropanol (Sigma-Aldrich, #I9516) for 10 minutes at room temperature. Pelleted RNA was washed with 70 % ethanol and solved in RNase‐free double-distilled water (ddH₂O). The transcript abundance of icaA_pAF, icaR_pAF and icaA_RN from three independent experiments was determined by real-time qRT-PCR. Thus, 5 µg of each RNA sample was treated with DNaseI (Thermofisher, #AM2235) for 45 min at 37°C and the reaction was stopped by phenol/chloroform/isoamylalcohol extraction (25:24:1, Carl Roth GmbH, #X985.2) with the aid of PLG heavy tubes (5 Prime, #2302830). RNA was precipitated overnight at -20°C with 4.67x volume ethanol/3M sodium acetate pH 6.5 (Thermofisher, #AM9740) (30:1 mix). Pelleted RNA was washed with 70% ethanol, dissolved in 30 µl RNase‐free ddH₂O and diluted 1:10. To check for efficiency of DNA digestion, a PCR was set up with the same amount of RNA (1 µl of 1:10) and same primers used for qRT-PCR. One-step qRT–PCR was performed using an amplification kit with SYBR Green (Power SYBR™, Green RNA-to-CT™ 1-Step Kit; Thermofisher, # 4389986) with the primers listed in Table 2 and run on Biorad CFX according to the manufacturer’s instructions. Transcript abundance was calculated using a logarithmic dilution series of one sample to generate a standard curve for each gene. Relative quantification of the genes of interest was expressed in relation to the expression of the constitutive reference gene gyrase B (gyrB). The means were calculated from three biological replicates run in technical duplicates. Statistical analysis was performed using one-way ANOVA by employing the GraphPad Prism software package.

Biofilm Assay

Biofilm formation was tested on 96-well, polystyrene tissue culture plates (Greiner Bio-One, # 655180) as described previously (Christensen et al., 1985), using Trypticase™ Soy Broth (BD BBL™, #211768) supplemented with 4% NaCl as growth medium. S. epidermidis RP62A and S. carnosus TM300 were used as positive and negative controls, respectively. For strains carrying resistance genes, antimicrobial agents were used at the following concentrations: 25 µg ml⁻¹ erythromycin (for both overnight and day culture) and 30 µg ml^-1 (for overnight culture) or 10 µg ml^-1 (for day culture) chloramphenicol. Bacterial overnight cultures were freshly diluted to OD₆₀₀ of 0.05 and 200 µl filled in each well (two technical replicates per strain). To distinguish between total, protein and PIA matrix-mediated biofilm production, three tissue culture plates were set up in parallel and incubated at 30°C for 18 h. Cultures were then discarded and adherent cells washed twice with 1x PBS buffer. The control plate for measuring the total biofilm was dried and heat-fixed at 65°C for 1 h. To discern between PIA- and protein- mediated biofilm, biofilms were either treated with 1 mg ml⁻¹ proteinase K (Merck, #1245680500) for 4 h at 37°C or 40 mM NaIO₄ (Carl Roth GmbH, #2603.1) for 24 h at 4°C. Afterwards the plates were washed with 1x PBS, dried and heat-fixed. All three plates were stained with 10 mg ml⁻¹ crystal violet (Merck, #115940) for 2 min, washed twice with double-distilled water before measuring the absorbance at 492 nm (ELISA plate reader, Multiskan Ascent). The means were calculated from three biological replicates. Statistical analysis was performed using one-way ANOVA by employing the GraphPad Prism software package.

Results

The Two ica Locus Copies in S. aureus Rd11 Are of Different Genetic Origin

We previously reported that database searches against the entire non-redundant nucleotide collection at NCBI (including whole-genome shotgun contigs) returned similarities of pAFS11 to an ica-like gene cluster present in some S. sciuri isolates [now M. sciuri (Madhaiyan et al., 2020)] (Feßler et al., 2017). Of note, the putative ica locus on pAFS11 was found to differ on nucleotide level from ica sequences present in S. aureus and S. epidermidis (Feßler et al., 2017). For further phylogenetic analysis, we therefore performed multiple sequence alignments of icaR/icaADBC nucleotide sequences from two S. aureus (i.e. RN4220 and ST398, to which Rd11 belongs) and two S. epidermidis (i.e. RP62A and O-47) strains as well as from the pAFS11 ica locus (referred to as ica_pAF hereafter). Finally, based on the nucleotide BLAST query results, an ica-like locus from the CoNS species S. sciuri was included into the analysis as well. Average distances were calculated from the alignment data and the tree displayed in Figure 1A illustrates that ica_pAF is most distantly related to the two S. aureus-derived ica loci. In addition, the two ica loci from S. epidermidis are highly divergent from ica_pAF, while they are closer related to ica from S. aureus. Interestingly, however, the ica_pAF nucleotide sequence is closely related to the ica-like locus from S. sciuri strain NS1 (Figures 1A, B). The data suggest that the two ica loci present in Rd11 are of different genetic origin, with ica_pAF most likely being derived from another species for which S. sciuri (M. sciuri) is a putative candidate. Although, as expected, interspecies conservation on the nucleotide level was found to be low (Figure 1B), the ica_pAF genes translate into amino acid sequences that are identified by the BLASTP algorithm as Ica-associated proteins with (again) some sequence differences between species. Thus, Figure 1C shows the percentage of identical amino acid positions upon pairwise alignments of Ica proteins from ica_pAF, S. aureus RN4220 and S. epidermidis RP62A. While comparisons between RN4220 and RP62A revealed high conservation of S. aureus and S. epidermidis Ica proteins, identical amino acid positions were much lower between ica_pAF and S. aureus-derived Ica proteins. In this respect, IcaR was the protein with the lowest conservation (29%), indicating that the two IcaR repressor proteins harbored by S. aureus Rd11 differ significantly on the protein sequence level (Figure 1C). Despite this apparent divergence, IcaR_pAF exhibits a number of amino acid residues (marked by asterisks in the conservation histogram in Figure 1D) that are highly conserved in IcaR proteins of S. aureus and S. epidermidis as well (Figure 1D). These include the putative icaA operator-interacting residues shown for IcaR S. epidermidis (Jeng et al., 2008) (marked with red triangles on top of the sequence in Figure 1D).

Plasmid pAFS11 Has a Negative Effect on S. aureus PIA-Mediated Biofilm Formation

As mentioned above, we previously reported that S. aureus Rd11 carrying pAFS11 does not produce biofilm, nor does S. aureus RN4220 into which the plasmid was transformed, suggesting that ica_pAF on the plasmid might be inactive (Feßler et al., 2017). To challenge this hypothesis, we cloned the entire ica_pAF from pAFS11 onto the shuttle vector pRB473 (resulting in plasmid pica_pAF) to enable ready genetic manipulation of the locus. As a first step, we deleted the icaR_pAF coding region from the vector, yielding plasmid pica_pAF_ΔicaR. Both plasmids (with or without icaR_pAF) were transformed into S. aureus RN4220 as recipient strain and biofilm assays were performed with the constructs and corresponding wild types (Figure 2). The biofilm assays allow to detect total biofilm formation as well as to differentiate between PIA and protein matrix-mediated biofilm production (see material and methods for details). We display here (and in the following figures) solely the data for PIA-mediated biofilm formation (Figures 2–5). The entire data sets on total, protein and PIA biofilm formation can be found in Supplementary Figure S1. The assays confirmed the PIA biofilm-negative phenotype of S. aureus Rd11 and revealed that the S. aureus RN4220 wild type is a weak, but detectable PIA biofilm producer. Upon acquisition of pAFS11, PIA biofilm formation of RN4220 did not increase, but on the contrary was even slightly reduced, although this reduction was statistically not significant (+pAFS11, Figure 2). The same phenomenon occurred when S. aureus RN4220 was transformed with plasmid pica_pAF, carrying the entire ica_pAF locus from pAFS11 (+pica_pAF, Figure 2). In contrast, however, PIA biofilm formation of RN4220 massively increased when the icaR_pAF gene was deleted from the ica_pAF copy on the vector (+pica_pAF_ΔicaR, Figure 2). In the S. aureus RN4220 +pica_pAF_ΔicaR strain, PIA biofilm levels even exceeded that of the S. epidermidis RP62A positive control and were much higher than in the RN4220 wild type, suggesting that the ica_pAF copy on the vector contributes to PIA production, but only when the IcaR_pAF repressor is absent. We conclude from this that pAFS11 may exert its negative effect on S. aureus PIA biofilm formation most likely via IcaR_pAF which also seems to negatively influence the ica locus on the RN4220 chromosome (hereinafter referred to as ica_RN).

FIGURE 2

Figure 2 Effect of pAFS11 on PIA biofilm formation. Analysis of PIA biofilm production by static 96-well microtiter plate biofilm assays of Rd11, RN4220 and RN4220 transformed with plasmid pAFS11 or with a plasmid carrying the whole ica operon from pAFS11 (+pica_pAF) or with icaR deletion (+pica_pAF_ΔicaR). RP62A served as positive control, TM300 as negative control. The means were calculated from three biological replicates run in duplicates. The ica genes distinctive for each strain are depicted as symbols, with filled symbols indicating presence and empty symbols indicating absence of a given gene (as indicated in the legend). The entire data sets on total, protein and PIA biofilm formation can be found in Supplementary Figure S1. Statistical analysis was performed using one-way ANOVA by employing the GraphPad Prism software package. ns: P = 0.1234; ****P < 0.0001.

The ica Locus on pAFS11 Is Inactive Due to Efficient IcaR Repression

The data obtained so far strongly suggest that the ica_pAF locus is functional and capable to enable PIA synthesis. However, by our initial experimental set-up (i.e. by employing the S. aureus RN4220 wild type with an intact chromosomal ica_RN locus) it was difficult to distinguish between ica_pAF- and ica_RN-derived PIA production. Therefore, we constructed a markerless ica_RN deletion mutant in RN4220 via allelic replacement, and transformed the resulting RN4220 Δica strain with plasmids pAFS11, pica_pAF and pica_pAF_ΔicaR. Biofilm assays with the constructs confirmed loss of PIA production in the RN4220 Δica deletion mutant (Figure 3A). Providing the mutant with an entire ica_pAF locus either on plasmid pAFS11 or pica_pAF did not result in biofilm formation (Figure 3A). However, biofilm formation was triggered and highly significantly increased, when the icaR_pAF repressor-encoding gene was deleted from the ica_pAF locus (+pica_pAF_ΔicaR, Figure 3A), indicating that the icaADBC_pAF genes of ica_pAF are indeed able to mediate PIA biofilm formation, once IcaR_pAF-dependent repression is alleviated. To further corroborate this assumption, we monitored transcription of icaA_pAF and icaR_pAF by qRT-PCR in the various constructs. In strain RN4220 Δica, transformed with either pAFS11 or pica_pAF, weak icaA_pAF transcription was detectable (Figure 3B). Upon deletion of icaR_pAF from the plasmid (+pica_pAF_ΔicaR, Figure 3B), icaR_pAF transcription was no longer detectable (as expected) and icaA_pAF transcription levels massively increased (i.e. 200-fold) compared to the intact ica_pAF copy (+pica_pAF, Figure 3B). These findings are in agreement with the biofilm test results. From the combined data we conclude that (i) the ica_pAF locus on pAFS11 is functionally fully intact and (ii) icaADBC_pAF operon transcription is efficiently repressed by its cognate IcaR_pAF repressor.

FIGURE 3

Figure 3 The ica genes of pAFS11 lead to biofilm formation. (A) Analysis of PIA biofilm production by static 96-well microtiter plate biofilm assays of strain RN4220 wild type and Δica alone or complemented with plasmid pAFS11 or with a plasmid carrying the whole ica operon from pAFS11 (+pica_pAF) or with icaR deletion (+pica_pAF_ΔicaR). RP62A served as positive control, TM300 as negative control. The entire data sets on total, protein and PIA biofilm formation can be found in Supplementary Figure S1. (B) Quantification of icaA_pAF and icaR_pAF transcripts by qRT-PCR of strains from (A). The graph displays relative mRNA amounts using gyrB expression as reference. (A, B) The ica genes distinctive for each strain are depicted as symbols, with filled symbols indicating presence and empty symbols indicating absence of a given gene (as indicated in the legend). The means were calculated from three biological replicates run in duplicates. Statistical analysis was performed using one-way ANOVA by employing the GraphPad Prism software package. ns: P = 0.1234; ****P < 0.0001.

IcaR From pAFS11 Represses the ica Locus in the S. aureus Chromosomal DNA and vice versa

Our initial experiments with wild type S. aureus RN4220 indicated that IcaR_pAF may also inhibit icaADBC_RN expression on the RN4220 chromosome (Figure 2). To substantiate this hypothesis, we constructed another set of plasmids carrying (i) the ica locus from RN4220 (pica_RN), (ii) the ica locus from RN4220 lacking icaR_RN (pica_RN_ΔicaR_RN) and (iii) the ica locus from RN4220 where we exchanged icaR_RN from RN4220 with icaR_pAF from pAFS11 (pica_RN_ΔicaR_RN_icaR_pAF). All plasmids were transformed into the RN4220 Δica mutant background and the resulting strains were analyzed for their ability to form PIA biofilm (Figure 4A). Complementation of RN4220 Δica with its own ica_RN locus restored PIA-mediated biofilm formation, and upon icaR_RN repressor gene deletion, PIA biofilm production significantly increased, demonstrating functionality of the vector-borne ica_RN locus, including icaR_RN-mediated regulation (Figure 4A). Accordingly, qRT-PCR analysis confirmed that IcaR_RN efficiently represses transcription of its cognate icaADBC_RN operon (Figure 4B). We then asked the question whether or not expression of the chromosomal icaADBC_RN operon can undergo control by the foreign IcaR_pAF repressor from pAFS11. Thus, we performed biofilm tests and quantitative transcription analyses with vector pica_RN_ΔicaR_icaR_pAF, in which icaADBC_RN was combined with the icaR_pAF gene from pAFS11. As shown in Figure 4, presence of icaR_pAF significantly diminished PIA production and transcription of the icaADBC_RN operon, suggesting the capability of IcaR_pAF to control the icaADBC_RN copy from RN4220 (Figure 4). Vice versa, we next investigated, if IcaR_RN from RN4220 can influence icaADBC_pAF from pAFS11. For this purpose, we additionally constructed vector pica_pAF_ΔicaR_pAF_icaR_RN which was transformed into the RN4220 Δica mutant background. Biofilm testing revealed a highly significant reduction of PIA production when icaADBC_pAF was combined with icaR_RN, suggesting that IcaR from S. aureus RN4220 is indeed able to repress the icaADBC_pAF genes from pAFS11 (Figure 4C).

FIGURE 4

Figure 4 IcaR_pAF controls expression of ica genes from RN4220 and vice versa. (A) Analysis of PIA biofilm production by static 96-well microtiter plate biofilm assays of strain RN4220 wild type and Δica alone or complemented with a plasmid carrying the ica operon of RN4220 (+pica_RN) or with icaR deletion (+pica_RN_ΔicaR) or with icaR_pAF instead of icaR_RN(+pica_RN_ΔicaR_icaR_pAF). The entire data sets on total, protein and PIA biofilm formation can be found in Supplementary Figure S1. (B) Quantification of icaA_RN and icaR_pAF transcripts by qRT-PCR of complemented strains from (A). The graph displays relative mRNA amounts using gyrB expression as reference. (C) Analysis of PIA biofilm production by static 96-well microtiter plate biofilm assays of RN4220 wild type and Δica alone or complemented with the ica operon of plasmid pAFS11 on a working plasmid (+pica_pAF) or the ica operon of plasmid pAFS11 with icaR deletion (+pica_pAF_ΔicaR) or with icaR_RN instead of icaR_pAF (+pica_pAF_ΔicaR_icaR_RN). (A, C) RP62A served as positive control, TM300 as negative control. (A–C) The ica genes distinctive for each strain are depicted as symbols, with filled symbols indicating presence and empty symbols indicating absence of a given gene (as indicated in the legend). The means were calculated from three biological replicates run in duplicates. Statistical analysis was performed using one-way ANOVA by employing the GraphPad Prism software package. ns: P = 0.1234; *P = 0.0332; ****P < 0.0001.

A Palindrome Sequence in the icaA_pAF Upstream Region Is Required for IcaR-Mediated Biofilm Repression

The data obtained so far demonstrate that the pAFS11- and RN4220-derived IcaR repressors, which differ on amino acid level (Figure 1D), are able to control the icaADBC operon of the respective other ica locus copy. To understand the molecular prerequisites for the IcaR interactions with their DNA targets, we focused on the nucleotide sequence constraints known to be involved in IcaR binding. IcaR was previously shown to bind as a dimer to a specific palindrome sequence (ACCTANCTNNC/GNNAGNTAGGT) present in the icaA operator of S. epidermidis (Jeng et al., 2008). This palindrome is 22 nucleotides long and contains six mismatches (22,6) (Figure 5A, top). Of note, the sequence is highly conserved and is also present in the S. aureus icaA promoter region (Figure 5A, top). Surprisingly, although IcaR from S. aureus is clearly able to control PIA production from pAFS11, the S. aureus-like palindrome sequence stretch lacks in ica_pAF (Figure 5A, bottom). Instead, the icaA_pAF upstream region displays two other palindromes which differ at the nucleotide level from that of the known S. aureus/S. epidermidis recognition site. Thus, palindrome A (TNAAAATNNTA/TANNATTTTNA) is 22 nucleotides long and harbors six mismatches (22,6), while palindrome B (CNAACNANC/GNTNGTTNG) consists of 18 nucleotides with six mismatches (18,6) (Figure 5A, bottom). To investigate the putative involvement of these palindromes in IcaR function, we mutated either palindrome A or B in (i) a plasmid carrying the whole ica_pAF, (ii) in an ica_pAF plasmid with an icaR_pAF deletion as well as (iii) in an icaR_pAF deletion vector complemented with icaR_RN. The plasmids were again transformed into the RN4220 Δica mutant background and analyzed for PIA-mediated biofilm formation. Figure 5B demonstrates that an altered palindrome A sequence did not influence icaR-mediated biofilm control. Thus, upon palindrome A mutation, PIA-mediated biofilm production remained repressed in pica_pAF(A***₎, indicating that IcaR_pAF does not require this nucleotide sequence stretch for action. As expected, PIA production was derepressed when icaR_pAF was lacking in the palindrome A mutant (+pica_pAF(A***₎_ΔicaR, Figure 5B). Most importantly, however, icaR_RN was still able to completely downregulate PIA-mediated biofilm production in this construct, confirming that palindrome A is not an interaction site for IcaR, neither for IcaR proteins derived from pAFS11 nor from RN4220 (Figure 5B). In contrast, mutation of palindrome B had a profound impact on biofilm regulation by IcaR. Firstly, PIA production was found to be deregulated and increased in an ica_pAF construct carrying an altered palindrome B nucleotide sequence (+pica_pAF(B***₎), suggesting that control by the cognate IcaR_pAF is significantly impaired when integrity of this sequence stretch is disturbed (Figure 5B). Moreover, PIA-mediated biofilm production further increased in a palindrome B mutant in which icaR_pAF was deleted (+pica_pAF(B***₎_ΔicaR, Figure 5B), which speaks in favor of residual IcaR_pAF repressor activity in the palindrome B mutant. Finally, when providing the mutant with IcaR_RN from RN4220, biofilm repression was partially, but not fully restored which (again) is in good agreement with residual IcaR repression in the palindrome B mutant. Together, the combined data strongly suggest that IcaR from both pAFS11 and RN4220 require an intact palindrome B (but not palindrome A) for unfolding their repressor activity on the ica_pAF locus.

FIGURE 5

Figure 5 IcaR proteins from S. aureus and from pAFS11 require a palindrome on the icaA_pAF operator for action. (A) TOP: Known palindrome sequence on S. aureus/S. epidermidis is shown at the top of the panel (Jeng et al., 2008). 22,6 stands for a palindrome which is 22 nt long and carries six mismatches. BOTTOM: Putative palindromes A and B on icaA_pAF operator are shown and their characteristics are reported. The nucleotide mutations introduced are shown in red below the wild type sequence alongside with the resulting palindrome perturbations. (B) Analysis of PIA biofilm production by static 96-well microtiter plate biofilm assays of strain RN4220 Δica complemented with the ica operon from pAFS11 wild type (+pica_pAF) or with deletion of icaR_pAF (+pica_pAF_ΔicaR) or with icaR_RN instead of icaR_pAF (+pica_pAF_ΔicaR_icaR_RN), carrying a mutated palindrome A (ica_pAF(A***₎) or B (ica_pAF(B***₎). The ica genes distinctive for each strains are depicted as symbols, with filled symbols indicating presence and empty symbols indicating absence of a given gene (as indicated in the legend). The entire data sets on total, protein and PIA biofilm formation can be found in Supplementary Figure S1. The means were calculated from three biological replicates run in duplicates. Statistical analysis was performed using one-way ANOVA by employing the GraphPad Prism software package. ns: P = 0.1234; *P = 0.0332; ****P < 0.0001.

Discussion

Acquisition of mobile genetic elements (MGEs) is often beneficial for bacteria by providing novel metabolic and resistance traits. However, MGE carriage may also come at considerable cost for the recipient bacterial cell (Slater et al., 2008; San Millan and MacLean, 2017). Thus, resources will be required to replicate and maintain MGEs (e.g. plasmids) on which beneficial genes are located and their (inappropriate) expression may impose a metabolic burden, resulting in reduced fitness and competitiveness of MGE-bearing bacterial cells (Baltrus, 2013). Accordingly, bacteria have evolved sophisticated mechanisms to control both MGE uptake and maintenance as well as expression of horizontally acquired genes (Brantl, 2014; Kwong et al., 2017; Firth et al., 2018). In case of plasmid-mediated HGT, this often involves a regulatory crosstalk between chromosomal factors and the newly acquired plasmid (Huang et al., 1990; Charles and Nester, 1993; Baños et al., 2009). Interestingly, these control networks are not unidirectional and there is growing evidence to suggest that plasmids are able to influence chromosomal gene expression as well in a wide range of bacterial species [recently reviewed in (Vial and Hommais, 2020)]. In the study presented here, we extend these examples by the Gram-positive pathogen S. aureus and reveal that horizontally acquired and core genome genes have the capacity to mutually influence each other in this organism. Thus, we demonstrate that a transcription factor (i.e. IcaR), located on a large multi-resistance plasmid, is able to target a pathogenicity factor (i.e. icaADBC-mediated PIA biofilm formation) from the S. aureus core genome. Vice versa, the IcaR homolog from the S. aureus core genome was found to be able to silence transcription of plasmid-borne icaADBC genes, creating a bi-directional regulatory crosstalk between plasmid- and chromosomally encoded factors that eventually hindered metabolically costly PIA-mediated biofilm formation.

PIA consists of N-acetyl-glucosamines (GlcNac) molecules, and ample sugar and energy supplies are fuelled into GlcNac synthesis to provide the building blocks of the exopolysaccharide. Accordingly, ica gene expression is intimately linked to central carbon flux control and energy balance (Vuong et al., 2005; Seidl et al., 2008; Zhu et al., 2009; Sadykov et al., 2011; Lindgren et al., 2014) which also involves the action of non-coding RNAs to appropriately adjust metabolic patterns (Rochat et al., 2018; Bronesky et al., 2019; Marincola et al., 2019; Schoenfelder et al., 2019). Presence of two fully functional ica gene clusters in strain S. aureus Rd11 is likely to represent a major metabolic challenge and the observed downregulation of PIA production in this strain makes sense to prevent metabolic overload. Paradoxically, it is just the additionally acquired ica_pAF locus copy on plasmid pAFS11 that mediates a biofilm-negative phenotype. Indeed, acquisition of pAFS11 or the ica_pAF locus alone abolished PIA biofilm formation in S. aureus (Figure 2). This effect is accomplished through the IcaR_pAF repressor which can also target the chromosomal ica locus copy (Figure 4). Moreover, the tight self-control of the ica_pAF copy on pAFS11 by its cognate IcaR_pAF repressor further contributes to a biofilm-negative phenotype (Figures 2, 3). Remarkably, icaADBC_pAF expression can be additionally repressed by Ica_RN from the core genome (Figure 4C). Thus, although being different from the canonical IcaR recognition site known from S. epidermidis and S. aureus (Jeng et al., 2008), Ica_RN interacts with a palindrome sequence present in the upstream region of icaADBC_pAF (palindrome B in Figure 5A), revealing a certain flexibility of IcaR-like proteins in DNA target selection (Figure 5).

Phylogenetic analyses revealed that the two ica loci in Rd11 differ both on nucleotide and protein sequence levels and are only distantly related to each other (Figure 1). Thus, the horizontally acquired ica_pAF copy on pAFS11 is likely to originate from an unknown bacterium for which the soil and animal dwelling species S. sciuri [recently re-classified as M. sciuri (Madhaiyan et al., 2020)] might represent a putative candidate (Feßler et al., 2017). But clearly more detailed investigations will be required to substantiate this hypothesis. Interestingly, inhibition of core genome-encoded exopolysaccharide production by plasmids seems to be a common theme in the bacterial world. Thus, in nitrogen-fixing Rhizobium tropici, exopolysaccharide production was found to be downregulated by the NrcR transcription factor, encoded on an acquired plasmid (Del Cerro et al., 2016), and in the nosocomial pathogen Acinetobacter baumanii, PNAG (PIA) production was described to be diminished upon acquisition of a multi-resistance plasmid (Venanzio et al., 2019). In the latter case, this large conjugative multiresistance plasmid facilitates its own transmission by downregulating chromosomally encoded type-6-secretion systems (T6SS) that usually hamper HGT (Venanzio et al., 2019). Together with the effect on PNAG production, the A. baumanii plasmids represent interesting examples for a plasmid-chromosome regulatory crosstalk that influences simultaneously both virulence and resistance traits.

LA-MRSA lineages of the clonal complex CC398 (to which strain Rd11 belongs to) can thrive in very different habitats (e.g. animals, humans, environment etc.) where they are exposed to numerous stress conditions. It is tempting to speculate that PIA biofilm formation, which is an important factor in S. aureus pathogenesis and survival (Fluckiger et al., 2005; Kropec et al., 2005), might become a selection advantage at some stage. In this respect, it is an interesting question by which mechanism(s) biofilm formation could be restored in the Rd11 isolate. Plasmid pAFS11 displays a striking mosaic structure and has most likely arisen by recombination of genes from various origins. Assuming that ica_pAF locus integration into pAFS11 and acquisition of the plasmid by S. aureus is an evolutionarily recent event, it is conceivable that control of pAFS11-encoded gene expression is not fully integrated into the regulatory network of the S. aureus Rd11 recipient (yet). One obvious possibility to (re)gain a biofilm positive phenotype would be (spontaneous) mutation and inactivation of icaR_pAF on pAFS11. At first sight, this idea seems to be contradictory to our experimental findings showing that IcaR_RN has the potential to take over and replace the lacking IcaR_pAF activity. This mutual IcaR replacement seems to work particularly well, when both factors (icaR and icaADBC) are on the same replicon and are probably in an appropriate stoichiometric repressor/DNA equilibrium (Figure 4C). In agreement with this assumption, the IcaR_RN repressor effect was found to be less efficient when a single icaR_RN copy resides on the chromosome and icaADBC_pAF is located on a (multi-copy) plasmid (Figure 2). Here, icaR_pAF deletion on the vector enabled PIA production, and it is reasonable to suggest that the insufficient repressor activity of Ica_RN in this experimental set-up might be associated with the copy number of the ica_pAF-bearing vector which increased the number of DNA targets for the IcaR_RN repressor. At the present stage of experimental work this is mere speculation. However, recent research demonstrates that available IcaR protein amounts are critical for the appropriate control of icaADBC transcription, and multiple cis- and trans-acting factors have been identified that target icaR mRNA molecules to fine-tune their translation (Ruiz de los Mozos et al., 2013; Rochat et al., 2018; Bronesky et al., 2019; Lerch et al., 2019; Schoenfelder et al., 2019). Plasmid copy number effects may have the potential to interfere with this delicate repressor/DNA target balance. In this respect, the data obtained with ica_pAF-bearing plasmids in the ica_RN locus proficient S. aureus RN4220 background may reflect very well the natural situation in pAFS11 carrying isolates (Figure 2). Thus, from an evolutionary point of view, mutational inactivation of icaR_pAF on the pAFS11 plasmid would make sense to readily enable PIA production. The same effect could be achieved by acquiring mutations in the ica_pAF palindrome B sequence, which represents the target site of both the IcaR_pAF and IcaR_RN proteins. Indeed, at least for S. aureus core genome ica loci, mutations in icaA upstream regions were previously described. The mutations had direct consequences for PIA production and occurred both in vitro and in vivo, indicating that the icaA promoter region undergoes selection and is a suitable target to modulate PIA production (Jefferson et al., 2003; Schwartbeck et al., 2016). It will be interesting to explore if the ica_pAF locus on pAFS11 might become subject to mutational variation. Long-term in vitro passage experiments together with surveillance of the evolution of pAFS11-like plasmids in field studies will be suitable approaches to give answers to how control of plasmid-borne ica locus expression will be integrated into the regulatory network of S. aureus.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Author Contributions

GM, AF, SS, and WZ: conceived and designed the experiments. GM, GJ and A-KK performed the experiments. GM, GJ, A-KK, FW and WZ analyzed the data. GM and WZ wrote the manuscript. All authors contributed to the article and approved the submitted version.

Funding

The study was supported by the German Research Council (DFG) through grant ZI665/3-1 as well as by the German Federal Ministry of Education and Research (BMBF), grant numbers 01KI1727E, 01KI1727D, 01KI2009D as part of the Research Network Zoonotic Infectious Diseases as well as grant number 16GW0297 within the program ‘Target validation for pharmaceutical drug development’. This publication was supported by the Open Access Publication Fund of the University of Wuerzburg.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We thank Dr. Sonja Schoenfelder for critical discussions and her continuous interest in this project.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2021.660702/full#supplementary-material

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