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Main conclusion PvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis, which rely on the phosphorylation status of T589, the endocytic YXXØ motif and the kinase activity of the receptor. Abstract Legume-rhizobia nodulation is a complex developmental process. It initiates when the rhizobia-produced Nod factors are perceived by specific LysM receptors present in the root hair apical membrane. Consequently, SYMRK (Symbiosis Receptor-like Kinase) becomes active in the root hair and triggers an extensive signaling network essential for the infection process and nodule organogenesis. Despite its relevant functions, the underlying cellular mechanisms involved in SYMRK signaling activity remain poorly characterized. In this study, we demonstrated that PvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis. We found that in uninoculated roots, PvSYMRK-EGFP is mainly associated with the plasma membrane, although intracellular puncta labelled with PvSymRK-EGFP were also observed in root hair and nonhair-epidermal cells. Inoculation with Rhizobium etli producing Nod factors induces in the root hair a redistribution of PvSYMRK-EGFP from the plasma membrane to intracellular puncta. In accordance, deletion of the endocytic motif YXXØ (YKTL) and treatment with the endocytosis inhibitors ikarugamycin (IKA) and tyrphostin A23 (TyrA23), as well as brefeldin A (BFA), drastically reduced the density of intracellular PvSYMRK-EGFP puncta. A similar effect was observed in the phosphorylation-deficient (T589A) and kinase-dead (K618E) mutants of PvSYMRK-EGFP, implying these structural features are positive regulators of PvSYMRK-EGFP endocytosis. Our findings lead us to postulate that rhizobia-induced endocytosis of SYMRK modulates the duration and amplitude of the SYMRK-dependent signaling pathway.

Xyloglucan endotransglucosylase/hydrolases (XTHs) are a glycoside hydrolase protein family involved in the biosynthesis of xyloglucans, with essential roles in the regulation of plant cell wall extensibility. By taking advantage of the whole genome sequence in Solanum lycopersicum, 37 SlXTHs were identified in the present work. SlXTHs were classified into four subfamilies (ancestral, I/II, III-A, III-B) when aligned to XTHs of other plant species. Gene structure and conserved motifs showed similar compositions in each subfamily. Segmental duplication was the primary mechanism accounting for the expansion of SlXTH genes. In silico expression analysis showed that SlXTH genes exhibited differential expression in several tissues. GO analysis and 3D protein structure indicated that all 37 SlXTHs participate in cell wall biogenesis and xyloglucan metabolism. Promoter analysis revealed that some SlXTHs have MeJA- and stress-responsive elements. qRT-PCR expression analysis of nine SlXTHs in leaves and roots of mycorrhizal colonized vs. non-colonized plants showed that eight of these genes were differentially expressed in leaves and four in roots, suggesting that SlXTHs might play roles in plant defense induced by arbuscular mycorrhiza. Our results provide valuable insight into the function of XTHs in S. lycopersicum, in addition to the response of plants to mycorrhizal colonization.

Legumes establish a symbiotic relationship with N2-fixing soil bacteria known as rhizobia. This process is initiated by a molecular dialog between roots and bacteria. Rhizobia secrete lipochitooligosaccharide signals, known as Nod factors, into the rhizosphere that are perceived by root hairs and induce an increase in Ca2+ levels, ROS production, and transcriptional activation of nodulin genes. Plant annexins are a family of calcium- and membrane-binding proteins involved in growth and development and in several abiotic and biotic responses. In this study, we identified thirteen annexin genes in the Phaseolus vulgaris (common bean) genome. We show that PvAnn1 transcript abundance was higher in root hairs than in root apices and stripped roots, and that during symbiosis, PvAnn1 transcript abundance was substantially higher in nodules than in nodule-stripped roots. RNAi knock-down of PvAnn1 resulted in reduced root hair lengths and ROS production. Furthermore, PvAnn1-RNAi transgenic roots that were inoculated with rhizobia showed a decrease in the progression and number of infection threads, possibly due to changes in Ca2+-ROS homeostasis and nodulin signaling. Furthermore, a reduced number of nodules and less nitrogen fixation was observed in the transgenic plants at 21 days post infection. Together, these data suggest that PvAnn1 is involved in rhizobial infection and nodule development and function.

In the Staphylococcus aureus ATCC25923 strain, the flqB mutation in the 5′untranslated region (5′UTR) of the norA gene causes increased norA mRNA expression and high efflux activity (HEA). The involvement of the norA gene 5′UTR in HEA has not been explored in S. epidermidis ; therefore, we examined the function of this region in S. epidermidis clinical isolates. The selection of isolates with HEA was performed based on ethidium bromide (EtBr) MIC values and efflux efficiency (EF) using the semi-automated fluorometric method. The function of the 5′UTR was studied by quantifying the levels of norA expression (RT-qPCR) and by identifying 5′UTR mutations by sequence analysis. Only 10 isolates from a total of 165 (6.1%) had HEA (EtBr MIC = 300 μg/ml and EF ranged from 48.4 to 97.2%). Eight of 10 isolates with HEA had the 5′UTR 95 Δ G mutation. Isolates carrying the 95 Δ G mutation had higher levels of norA expression compared with those that did not. To corroborate that the 95 Δ G mutation is involved in HEA, a strain adapted to EtBr was obtained in vitro . This strain also presented the 95 Δ G mutation and had a high level of norA expression and EF, indicating that the 95 Δ G mutation is important for the HEA phenotype. The 95 Δ G mutation produces a different structure in the Shine-Dalgarno region, which may promote better translation of norA mRNA. To our knowledge, this is the first report to demonstrate the participation of the 5′UTR 95 Δ G mutation of the norA gene in the HEA phenotype of S. epidermidis isolates. Here, we propose that the efflux of EtBr is caused by an increment in the transcription and/or translation of the norA gene.

In the Staphylococcus aureus ATCC25923 strain, the flqB mutation in the 5'untranslated region (5'UTR) of the norA gene causes increased norA mRNA expression and high efflux activity (HEA). The involvement of the norA gene 5'UTR in HEA has not been explored in S. epidermidis; therefore, we examined the function of this region in S. epidermidis clinical isolates. The selection of isolates with HEA was performed based on ethidium bromide (EtBr) MIC values and efflux efficiency (EF) using the semi-automated fluorometric method. The function of the 5'UTR was studied by quantifying the levels of norA expression (RT-qPCR) and by identifying 5'UTR mutations by sequence analysis. Only 10 isolates from a total of 165 (6.1%) had HEA (EtBr MIC = 300 μg/ml and EF ranged from 48.4 to 97.2%). Eight of 10 isolates with HEA had the 5'UTR

Keratitis caused by Staphylococcus aureus often leads to Vascular Endothelial Growth Factor (VEGF)-dependent neovascularization, but contribution of peptidoglycan (PGN), muramyl dipeptide (MDP) and lipoteichoic acid (LTA) from S. aureus to VEGF-dependent neovascularization has not been well-studied. This work was focused on the analysis of S. aureus cell wall components in the production of VEGF family members (VEGF-A, VEGF-B, VEGF-C and VEGF-D) in ocular limbal fibroblasts. Primary culture of human limbal fibroblasts (PCHLFs) were stimulated with PGN, MDP, and LTA, and VEGF family; toll-like receptor 2 (TLR2), nucleotide-binding oligomerization domain 1 (NOD1), and NOD2 expression were determined by RT-PCR. Anti-TLR2 antibody, epidermal growth factor receptor (EGFR) signaling inhibitors (AG1478 and PD98059), and NFκB activation were used to analyze VEGF-A by ELISA. TLR2 and NOD1 expression were analyzed by flow cytometry. The stimulation of PCHLFs with PGN and MDP increased the levels of VEGF-A expression (mRNA and protein) in a time-dependent and dose-dependent manner. VEGF-B, VEGF-C and VEGF-D were expressed constitutively, and no further induction was observed in stimulated PCHLFs. LTA did not increase the expression levels of the VEGF family. TLR2 mRNA and protein were increased only when PCHLFs were stimulated with PGN. Treatment with an anti-TLR2 antibody blocked the interaction of PGN with the receptor, inhibiting VEGF-A over-expression; the presence of anti-TLR2 antibodies did not affect the over-production of VEGF-A after MDP treatment. PCHLFs stimulated with PGN and MDP, but not with LTA, activated NFκB. MDP stimulated the production of NOD1 and NOD2 mRNAs in a time-dependent and dose-dependent manner, and NOD2 protein was only increased by MDP. Treatment of PCHLFs with AG1478 and PD98059 inhibitors prior to stimulation with MDP resulted in the inhibition of VEGF-A over-production, compared with PCHLFs stimulated with MDP alone. Taken together, these results suggest that limbal fibroblasts produce VEGF-A through PGN-TLR2-NFκB and MDP-NOD2-EGFR.

Aims:Staphylococcus epidermidis is considered a commensal bacterium; however, it is frequently isolated from ocular infections showing a multidrug resistance. Ciprofloxacin-resistant strains have been isolated from ocular infections; however, resistance to quinolone, such as gatifloxacin and moxifloxacin, is not often studied, consequently the resistance mechanism is unknown. Our aim was to address the quinolone resistance and to explore the resistance mechanism in S. epidermidis strains isolated from ocular infections. Methods:S. epidermidis strains were isolated from patients with conjunctivitis (n = 23), endophthalmitis (n = 14) and corneal ulcers (n = 7). Minimum inhibition concentrations were determined by broth and agar dilution methods for moxifloxacin, gatifloxacin, balofloxacin, rufloxacin and pazufloxacin. Mutations were identified by sequencing the gyrA and parC genes, and their expression was determined by reverse transcriptase polymerase chain reaction. Results: We found that 13.6% (6/44) of the strains were quinolone resistant. In endophthalmitis, 21.4% were gatifloxacin, moxifloxacin and balofloxacin resistant. In corneal ulcers, 14.2, 14.2 and 28.5% were gatifloxacin, moxifloxacin and balofloxacin resistant, respectively, and in conjunctivitis only 4.3% were gatifloxacin resistant. The 6 strains with quinolone resistance showed mutations at Ser84Phe for the gyrA gene, and Ser80Phe for the parC gene. Gatifloxacin did not change the expression levels of gyrA and parC genes. Conclusion:S. epidermidis strains isolated from three ocular pathologies were gatifloxacin and moxifloxacin resistant due to mutations on the gyrA and parC genes.

In ocular infections (OIs) caused by Staphylococcus epidermidis, biofilms composed mainly of poly-N-acetylglucosamine (PNAG) have been widely studied, but PNAG-independent biofilms have not. Therefore, we searched for a relationship between the ica operon (involved in PNAG-biofilm) and the biochemical composition of biofilms in isolates from OI. Isolates from OI (n = 62), from healthy conjunctiva (HC; n = 45) and from healthy skin (HS; n = 53), were used to detect icaA and icaD genes, and the insertion sequence 256 (IS256) using PCR. The compositions of the biofilms were determined by treatment with NaIO4, proteinase K and DNase I. Multilocus sequence typing (MLST) was performed to characterize the isolates, and the expression of aap and embp genes was determined by real-time qPCR. A strong relationship between the icaA −/icaD −/IS256 − genotype and protein- or protein/extracellular DNA (eDNA)-biofilm composition was found in the isolates from OI (53.6 %), whereas the icaA +/icaD +/IS256 − genotype and carbohydrate-biofilm was most prevalent in isolates from HC (25 %) and HS (25 %). Isolates with an icaA −/icaD −/IS256 − genotype and protein-biofilm phenotype were predominantly of the ST2 lineage, while carbohydrate-biofilm-producing strains were mainly of the ST9 lineage. The protein-biofilm-producing strains had higher expression levels of aap gene than carbohydrate-biofilm-producing strains; while embp gene did not have the same pattern of expression. These results suggest that S. epidermidis strains with icaA− /icaD− /IS256 − genotype and protein- or protein/eDNA-biofilms have a stronger ability to establish in the eye than S. epidermidis strains with icaA+ /icaD+ /IS256 − genotype and PNAG-biofilms.

Staphylococcus aureus employs a heme sensing system (HssR–HssS) and a heme-regulated transporter efflux pump (HrtA–HrtB) to avoid the accumulation of heme, which is toxic at high concentrations. The detoxification system to heme has not been studied in Staphylococcus epidermidis . In this work, the hssR, hssS, hrtA, and hrtB genes were detected, and their expression when stimulated by hemin in S. epidermidis was explored. In silico genomic analyses exhibited that the genetic organization of the hssRS and hrtAB genes was identical in 11 Staphylococcus species analyzed, including S. epidermidis. Slight variations were found in their syntenic regions. The predicted secondary structure of HrtAB proteins from these species was almost identical to these of S. aureus. Additionally, hrtAB promoter sequences of some species were analyzed, and 1 or 2 different nucleotide substitutions were found in the downstream motif. Concentrations of hemin above 5 µmol/L inhibited S. epidermidis growth. However, S. epidermidis that was pre-exposed to a subinhibitory hemin concentration (1 µmol/L) was able to grow when inoculated into medium containing above 5 µmol/L hemin. The expression levels of hrtA and hrtB genes in S. epidermidis exhibited a significant difference when they were stimulated with hemin. Our results suggest that the HrtAB could be involved in hemin detoxification of S. epidermidis.

The infection frequency associated to bacterial conjunctivitis, corneal ulcers (CU), and endophthalmitis was studied along a five years period. The isolation and identification of microorganisms were performed by culture-based methods and biochemical test respectively. Also, a nested PCR to detect gram-negative and gram-positive bacteria in the clinical samples was assayed. Nested PCR was a more efficient method than culture to detect bacteria in the samples. The most frequently isolated species was Staphylococcus epidermidis, a bacterium commonly considered as a human saprophyte. The S. epidermidis strains from conjunctivitis, CU, and endophthalmitis exhibited 46, 33.9, and 34.1% of oxacilin-resistance respectively. A total of 28% of intermediate-vancomycin resistance (MIC = 8-16 microg/ml) was observed among S. epidermidis strain collection. The UPGMA cluster analysis of the multiresistance profile data of intermediate vancomycin-resistant S. epidermidis strains showed a high phenotypic diversity and no relationship between each group and their clinical origin. The biofilm formation capacity was broadly distributed (66%), particularly among intermediate-vancomycin strains (75%). In brief, S. epidermidis displayed a high diversity of antibiotic resistance profiles and biofilm formation capacity. These phenotypic traits could explain the high isolation frequency of S. epidermidis from ocular infections and oblige to review the saprophytic status of these bacteria.

Staphylococcus epidermidis is considered a commensal bacterium; however, it is frequently isolated from ocular infections showing a multidrug resistance. Ciprofloxacin-resistant strains have been isolated from ocular infections; however, resistance to quinolone, such as gatifloxacin and moxifloxacin, is not often studied, consequently the resistance mechanism is unknown. Our aim was to address the quinolone resistance and to explore the resistance mechanism in S. epidermidis strains isolated from ocular infections.S. epidermidis strains were isolated from patients with conjunctivitis (n = 23), endophthalmitis (n = 14) and corneal ulcers (n = 7). Minimum inhibition concentrations were determined by broth and agar dilution methods for moxifloxacin, gatifloxacin, balofloxacin, rufloxacin and pazufloxacin. Mutations were identified by sequencing the gyrA and parC genes, and their expression was determined by reverse transcriptase polymerase chain reaction.We found that 13.6% (6/44) of the strains were quinolone resistant. In endophthalmitis, 21.4% were gatifloxacin, moxifloxacin and balofloxacin resistant. In corneal ulcers, 14.2, 14.2 and 28.5% were gatifloxacin, moxifloxacin and balofloxacin resistant, respectively, and in conjunctivitis only 4.3% were gatifloxacin resistant. The 6 strains with quinolone resistance showed mutations at Ser84Phe for the gyrA gene, and Ser80Phe for the parC gene. Gatifloxacin did not change the expression levels of gyrA and parC genes.S. epidermidis strains isolated from three ocular pathologies were gatifloxacin and moxifloxacin resistant due to mutations on the gyrA and parC genes.