Diversity in regulatory regions of icaADBCR and fnbAB genes among Staphylococcus aureus strains isolated from periprosthetic joint infections
Authors: Morales Liliana , Echeverz Maite , Trobos Margarita , Solano Cristina , Lasa Iñigo 
Published: July 2020

&lt;p&gt;&lt;strong&gt;Introduction: &lt;/strong&gt;The ability of bacteria to colonize implant surfaces and tissues as a biofilm plays a relevant role in medical-device-associated infections. &lt;em&gt;Staphylococcus aureus&lt;/em&gt; strains can produce a biofilm matrix made of the poly-N-acetylglucosamine (PIA/PNAG) exopolysaccharide and/or proteins. PIA/PNAG is synthesised by enzymes encoded by the&lt;em&gt; icaADBC&lt;/em&gt; operon whose expression is repressed by the transcriptional regulator IcaR, while the protein-dependent biofilm is commonly associated to fibronectin-biding proteins, FnBPA and FnBPB, encoded by&lt;em&gt; fnbA&lt;/em&gt; and&lt;em&gt; fnbB&lt;/em&gt; genes. The aim of this work was to identify common genetic features in the regulatory regions of biofilm-related genes among clinical&lt;em&gt; S. aureus&lt;/em&gt; strains derived from periprosthetic joint infections (PJI). &amp;#160;&lt;/p&gt;
&lt;p&gt;&lt;strong&gt;Material and Methods:&lt;/strong&gt; Genomes of 45 &lt;em&gt;S. aureus&lt;/em&gt; strains from PJI were sequenced. Firstly, the sequence comprising the entire &lt;em&gt;icaADBC&lt;/em&gt; regulatory region (5&amp;#8217;UTR of &lt;em&gt;icaADBC&lt;/em&gt; and &lt;em&gt;icaR&lt;/em&gt;, the &lt;em&gt;icaR&lt;/em&gt; coding sequence and its 3&amp;#8217;UTR region) and secondly, the sequence of the promoter region of &lt;em&gt;fnbAB&lt;/em&gt; were compared to those of&lt;em&gt; S. aureus&lt;/em&gt; MW2 strain. Regulatory regions containing distinctive features were identified, fused to a reporter gene and introduced in a reference strain to analyze differences in gene expression.&lt;/p&gt;
&lt;p&gt;&lt;strong&gt;Results: &lt;/strong&gt;In the case of the&lt;em&gt; icaADBC&lt;/em&gt; operon, single nucleotide polymorphisms (SNPs) in the &lt;em&gt;icaADBC&lt;/em&gt; regulatory region allowed clustering of the strains in five groups from which a representative strain was chosen for further studies: &lt;em&gt;S. aureus&lt;/em&gt; MIC 6924 (20% of isolates), MIC 6934 (13%), MIC 6936 (7%), MIC 6948 (2%) and MIC 7018 (4%). Of note, MICs 6948 and 7018 contained mutations in the&lt;em&gt; icaR&lt;/em&gt; coding sequence. In this respect, a single nucleotide mutation in &lt;em&gt;icaR&lt;/em&gt; (Val176Glu) caused a significant increase in &lt;em&gt;icaADBC&lt;/em&gt; transcription and thus, in PIA/PNAG production and biofilm formation. In contrast, none of the rest of the SNPs found in the&lt;em&gt; icaADBC&lt;/em&gt; regulatory region modified the transcription levels of the reporter gene. With respect to fnBPA and fnBPB genes and in agreement with previous studies, 100% of the strains contained the&lt;em&gt; fnbA&lt;/em&gt; gene whereas only 69% contained the&lt;em&gt; fnbB&lt;/em&gt; gene. The promoter region of &lt;em&gt;fnbA&lt;/em&gt; was found to be highly conserved. SNPs in the promoter region of &lt;em&gt;fnbB&lt;/em&gt; allowed clustering the strains in five groups. From these, the most frequently identified pattern was represented by &lt;em&gt;S. aureus&lt;/em&gt; MIC 6948 (53%) and correlated with a lower level of reporter expression, whereas the group containing SNPs in the LexA binding sites was represented by MIC 7014 (4%) and correlated with higher expression levels.&lt;/p&gt;
&lt;p&gt;&lt;strong&gt;Conclusion: &amp;#160;&lt;/strong&gt;Our results suggest that&lt;em&gt; S. aureus&lt;/em&gt; isolates from periprosthetic joint infections do not share specific features in cis regulatory regions of&lt;em&gt; icaADBC&lt;/em&gt; and &lt;em&gt;fnbB&lt;/em&gt; genes that may help to predict a higher expression levels of biofilm matrix compounds.&lt;/p&gt;
&lt;p&gt;&lt;strong&gt;Acknowledgement: &lt;/strong&gt;This project has received funding from the European Union's H2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No 801586.&lt;/p&gt;