Susceptibilidad de Cepas de Candida Oral a Extracto Etanólico del Propóleo Chileno de Olmué / Susceptibility of Oral Candida Strains to Ethanol Extract of Chilean Propolis of Olmué

RESUMEN: La infección por Candida albicans en la mucosa oral es conocida como Candidiasis oral (CO) y se diagnostica por el reconocimiento de cambios clínicos y la presencia de pseudohifas, hifas o levaduras en muestras obtenidas por citología exfoliativa o biopsia. Los agentes farmacológicos tópicos clásicos para el tratamiento de CO son Nistatina y Miconazol. Sin embargo, a pesar de las distintas terapias contra CO, existen formas de Candida resistentes al tratamiento convencional. El objetivo de este estudio fue determinar la susceptibilidad in vitro de Candida spp. a un extracto etanólico de propóleo de Olmué. Se realizó un estudio experimental descriptivo in vitro en donde se evaluó el efecto que presenta el uso de extracto etanólico de propóleo como antifúngico sobre cepas de Candida spp. obtenidas de la cavidad oral (mucosa palatina) de 31 individuos, con candidiasis oral diagnosticados con estomatitis subprotésica. El propóleo chileno utilizado fue obtenido de la zona geográfica de Olmué, quinta región. Se encontró que el 100 % de las muestras en rangos de concentración de propóleo de 0,1 µg/mL y 1,6 µg/mL presentaron un grado de inhibición en el crecimiento de Candida Oral y por otra parte el extracto etanólico de propóleo que generó inhibición en la mayor cantidad de muestras fue al 0,4 µg/mL (41,94 % de las muestras) y en segundo lugar la concentración al 0,2 µg/mL (35 % de las muestras). Se concluyó que el extracto etanólico de propóleo chileno obtenido de la zona de Olmué presenta la capacidad de inhibir el crecimiento de Candida spp. en agar Sabouraud in vitro de forma dosis dependiente.

ABSTRACT: Fungal (or yeast) infections; mycoses, occurring in the oral mucous membranes, of Candida species (mostly C. albicans, a normal component of the oral microbiota), also known as oral thrush or oral candidiasis (OC), can be diagnosed via the recognition of clinical changes and the presence of pseudohyphae, hyphae or yeasts in samples obtained by exfoliative cytology and/or biopsy. Topical pharmacological preparations and drugs such as Nystatin and Miconazole are used in the treatment of CO. However, there are forms of Candida with resistance to such conventional treatment approaches. Therefore, the aim of this study is to determine the in vitro susceptibility of Candida spp.; an ethanolic extract of propolis from Olmué. Hence, an experimental in vitro descriptive study was carried out in which the effect of an ethanolic extract of propolis used as antifungal on strains of Candida spp. obtained from the oral cavity (palatine mucosa) of 31 individuals, diagnosed with OC (subdenture stomatitis) is determined. Natural propolis was obtained from the Olmué area, in the 5th region of Chile. It was found that 100 % of the samples with propolis concentration ranging from 0.1 mg / mL to 1.6 mg / mL presented a degree of inhibition in the growth of OC. On the other hand, the ethanolic extract of propolis that generated inhibition in the largestnumber of samples was 0.4 mg / mL (41.94 % of the samples) followed by the concentration of 0.2 mg / mL (35 % of the samples). Therefore, it can be concluded that the ethanolic extract of Chilean propolis obtained from the Olmué area has the ability to inhibit the growth of Candida spp. in vitro in a dosage-dependent manner.

Demonstration of the functional role of conserved Glu-Arg residues in the Staphylococcus aureus ferrichrome transporter.

The features that govern the interaction of ligand binding proteins with membrane permeases of cognate ABC transporters are largely unknown. Using sequence alignments and structural modeling based on the structure of the Escherichia coli BtuCD vitamin B12 transporter, we identified six conserved basic residues in the permease, comprised of FhuB and FhuG proteins, in the ferrichrome transporter of Staphylococcus aureus. Using alanine-scanning mutagenesis we demonstrate that two of these residues, FhuB Arg-71 and FhuG Arg-61, play a more dominant role in transporter function than FhuB Arg-74 and Arg-311, and FhuG Arg-64 and Lys-306. Moreover, we show that at positions 71 and 61 in FhuB and FhuG, respectively, arginine cannot be substituted for lysine without loss of transporter function. Previously, our laboratory demonstrated the importance of conserved acidic residues in the ferrichrome binding protein, FhuD2. Taken together, these results support the hypothesis that Glu-Arg salt bridges are critical for the interaction of the ligand binding protein with the transmembrane domains FhuB and FhuG. This hypothesis was further studied by "charge swapping" experiments whereby we constructed a S. aureus strain expressing FhuD2 with conserved residues Glu-97 and Glu-231 replaced by Arg and FhuB and FhuG with conserved basic residues Arg-71 and Arg-61, respectively, replaced by Glu. A strain containing this combination of substitutions restored partial function to the ferrichrome transporter. The results provide a direct demonstration of the functional importance of conserved basic residues on the extracellular surface of the ferrichrome permease in the Gram-positive bacterium S. aureus.

Isolation of a novel Aggregatibacter actinomycetemcomitans serotype b bacteriophage capable of lysing bacteria within a biofilm.

A bacteriophage specific for Aggregatibacter actinomycetemcomitans serotype b, able to kill the bacterium within a biofilm, was isolated. Random mutagenesis of this phage rendered a bacteriophage able to kill 99% of the bacteria within a biofilm. This is the first report of a biocontrol experiment against A. actinomycetemcomitans.

Toll-like receptor 2 ligands on the staphylococcal cell wall downregulate superantigen-induced T cell activation and prevent toxic shock syndrome.

Staphylococcal superantigens are pyrogenic exotoxins that cause massive T cell activation leading to toxic shock syndrome and death. Despite the strong adaptive immune response induced by these toxins, infections by superantigen-producing staphylococci are very common clinical events. We hypothesized that this may be partly a result of staphylococcal strains having developed strategies that downregulate the T cell response to these toxins. Here we show that the human interleukin-2 response to staphylococcal superantigens is inhibited by the simultaneous presence of bacteria. Such a downregulatory effect is the result of peptidoglycan-embedded molecules binding to Toll-like receptor 2 and inducing interleukin-10 production and apoptosis of antigen-presenting cells. We corroborated these findings in vivo by showing substantial prevention of mortality after simultaneous administration of staphylococcal enterotoxin B with either heat-killed staphylococci or Staphylococcus aureus peptidoglycan in mouse models of superantigen-induced toxic shock syndrome.

Characterization of staphyloferrin A biosynthetic and transport mutants in Staphylococcus aureus.

Iron is critical for virtually all forms of life. The production of high-affinity iron chelators, siderophores, and the subsequent uptake of iron-siderophore complexes are a common strategy employed by microorganisms to acquire iron. Staphylococcus aureus produces siderophores but genetic information underlying their synthesis and transport is limited. Previous work implicated the sbn operon in siderophore synthesis and the sirABC operon in uptake. Here we characterize a second siderophore biosynthetic locus in S. aureus; the locus consists of four genes (in strain Newman these open reading frames are designated NWMN_2079-2082) which, together, are responsible for the synthesis and export of staphyloferrin A, a polycarboxylate siderophore. While deletion of the NWMN_2079-2082 locus did not affect iron-restricted growth of S. aureus, strains bearing combined sbn and NWMN_2079-2082 locus deletions produced no detectable siderophore and demonstrated severely attenuated iron-restricted growth. Adjacent to NWMN_2079-2082 resides the htsABC operon, encoding an ABC transporter previously implicated in haem acquisition. We provide evidence here that HtsABC, along with the FhuC ATPase, is required for the uptake of staphyloferrin A. The crystal structure of apo-HtsA was determined and identified a large positively charged region in the substrate-binding pocket, in agreement with a role in binding of anionic staphyloferrin A.

Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systems.

The PglB oligosaccharyltransferase (OTase) of Campylobacter jejuni can be functionally expressed in Escherichia coli, and its relaxed oligosaccharide substrate specificity allows the transfer of different glycans from the lipid carrier undecaprenyl pyrophosphate to an acceptor protein. To investigate the substrate specificity of PglB, we tested the transfer of a set of lipid-linked polysaccharides in E. coli and Salmonella enterica serovar Typhimurium. A hexose linked to the C-6 of the monosaccharide at the reducing end did not inhibit the transfer of the O antigen to the acceptor protein. However, PglB required an acetamido group at the C-2. A model for the mechanism of PglB involving this functional group was proposed. Previous experiments have shown that eukaryotic OTases have the same requirement, suggesting that eukaryotic and prokaryotic OTases catalyze the transfer of oligosaccharides by a conserved mechanism. Moreover, we demonstrated the functional transfer of the C. jejuni glycosylation system into S. enterica. The elucidation of the mechanism of action and the substrate specificity of PglB represents the foundation for engineering glycoproteins that will have an impact on biotechnology.

Requirement of Staphylococcus aureus ATP-binding cassette-ATPase FhuC for iron-restricted growth and evidence that it functions with more than one iron transporter.

In Staphylococcus aureus, fhuCBG encodes an ATP-binding cassette (ABC) transporter that is required for the transport of iron(III)-hydroxamates; mutation of either fhuB or fhuG eliminates transport. In this paper, we describe construction and characterization of an S. aureus fhuCBG deletion strain. The delta fhuCBG::ermC mutation not only resulted in a strain that was incapable of growth on iron(III)-hydroxamates as a sole source of iron but also resulted in a strain which had a profound growth defect in iron-restricted laboratory media. The growth defect was not a result of the inability to transport iron(III)-hydroxamates since S. aureus fhuG::Tn917 and S. aureus fhuD1::Km fhuD2::Tet mutants, which are also unable to transport iron(III)-hydroxamates, do not have similar iron-restricted growth defects. Complementation experiments demonstrated that the growth defect of the delta fhuCBG::ermC mutant was the result of the inability to express FhuC and that this was the result of an inability to transport iron complexed to the S. aureus siderophore staphylobactin. Transport of iron(III)-staphylobactin is dependent upon SirA (binding protein), SirB (permease), and SirC (permease). S. aureus expressing FhuC with a Walker A K42N mutation could not utilize iron(III)-hydroxamates or iron(III)-staphylobactin as a sole source of iron, supporting the conclusion that FhuC, as expected, functions with FhuB, FhuG, and FhuD1 or FhuD2 to transport iron(III)-hydroxamates and is the "genetically unlinked" ABC-ATPase that functions with SirA, SirB, and SirC to transport iron(III)-staphylobactin. Finally, we demonstrated that the delta fhuCBG::ermC strain had decreased virulence in a murine kidney abscess model.

Defective O-antigen polymerization in tolA and pal mutants of Escherichia coli in response to extracytoplasmic stress.

We have previously shown that the TolA protein is required for the correct surface expression of the Escherichia coli O7 antigen lipopolysaccharide (LPS). In this work, delta tolA and delta pal mutants of E. coli K-12 W3110 were transformed with pMF19 (encoding a rhamnosyltransferase that reconstitutes the expression of O16-specific LPS), pWQ5 (encoding the Klebsiella pneumoniae O1 LPS gene cluster), or pWQ802 (encoding the genes necessary for the synthesis of Salmonella enterica O:54). Both DeltatolA and delta pal mutants exhibited reduced surface expression of O16 LPS as compared to parental W3110, but no significant differences were observed in the expression of K. pneumoniae O1 LPS and S. enterica O:54 LPS. Therefore, TolA and Pal are required for the correct surface expression of O antigens that are assembled in a wzy (polymerase)-dependent manner (like those of E. coli O7 and O16) but not for O antigens assembled by wzy-independent pathways (like K. pneumoniae O1 and S. enterica O:54). Furthermore, we show that the reduced surface expression of O16 LPS in delta tolA and delta pal mutants was associated with a partial defect in O-antigen polymerization and it was corrected by complementation with intact tolA and pal genes, respectively. Using derivatives of W3110 delta tolA and W3110 delta pal containing lacZ reporter fusions to fkpA and degP, we also demonstrate that the RpoE-mediated extracytoplasmic stress response is upregulated in these mutants. Moreover, an altered O16 polymerization was also detected under conditions that stimulate RpoE-mediated extracytoplasmic stress responses in tol+ and pal+ genetic backgrounds. A Wzy derivative with an epitope tag at the C-terminal end of the protein was stable in all the mutants, ruling out stress-mediated proteolysis of Wzy. We conclude that the absence of TolA and Pal elicits a sustained extracytoplasmic stress response that in turn reduces O-antigen polymerization but does not affect the stability of the Wzy O-antigen polymerase.