The hmuQ and hmuD Genes from Bradyrhizobium japonicum Encode Heme-Degrading Enzymes

Utilization of heme by bacteria as a nutritional iron source involves the transport of exogenous heme, followed by cleavage of the heme macrocycle to release iron. Bradyrhizobium japonicum can use heme as an iron source, but no heme-degrading oxygenase has been described. Here, bioinformatics analyses of the B. japonicum genome identified two paralogous genes renamed hmuQ (bll7075) and hmuD (bll7423) that encode proteins with weak similarity to the heme-degrading monooxygenase IsdG from Staphylococcus aureus. The hmuQ gene is clustered with known heme transport genes in the genome. Recombinant HmuQ bound heme with a K(d) value of 0.8 μM and showed spectral properties consistent with a heme oxygenase. In the presence of a reductant, HmuQ catalyzed the degradation of heme and the formation of biliverdin. The hmuQ and hmuD genes complemented a Corynebacterium ulcerans heme oxygenase mutant in trans for utilization of heme as the sole iron source for growth. Furthermore, homologs of hmuQ and hmuD were identified in many bacterial genera, and the recombinant homolog from Brucella melitensis bound heme and catalyzed its degradation. The findings show that hmuQ and hmuD encode heme oxygenases and indicate that the IsdG family of heme-degrading monooxygenases is not restricted to gram-positive pathogenic bacteria

Positive Control of Ferric Siderophore Receptor Gene Expression by the Irr Protein in Bradyrhizobium japonicum▿

Ferric siderophore receptors are components of high-affinity iron-chelate transport systems in gram-negative bacteria. The genes encoding these receptors are generally regulated by repression. Here, we show that the ferrichrome receptor gene bll4920 and four additional putative ferric siderophore receptor genes in Bradyrhizobium japonicum are positively controlled by the regulatory protein Irr, as observed by the low level of mRNA transcripts in an irr mutant in iron-limited cells. Potential Irr binding sites with iron control element (ICE)-like motifs were found upstream and distal to the transcription start sites of the five receptor genes. However, purified recombinant Irr bound only some of those elements. Nevertheless, dissection of the bll4920 promoter region showed that a component in extracts of wild-type cells grown in iron-limited media bound only in the ICE motif region of the promoter. This binding was not observed with extracts of cells from the parent strain grown under high-iron conditions or from an irr mutant strain. Furthermore, gel mobility supershift experiments identified Irr as the binding protein in cell extracts. Chromatin immunoprecipitation experiments demonstrated that Irr occupies the promoters of the five ferric iron transport genes in vivo. We conclude that Irr is a direct positive regulator of ferric iron transport in B. japonicum

Structural Characterization of Histatin 5-Spermidine Conjugates : A Combined Experimental and Theoretical Study

Histatin 5 (Hst5) is a naturally occurring antimicrobial peptide that acts as the first line of defense against oral candidiasis. It has been shown that conjugation of the active Hst5 fragment, Hst54-15, and the polyamine spermidine (Spd) improves the candidacidal effect. Knowledge about the structure of these conjugates is, however, very limited. Thus, the aim of this study was to characterize the structural properties of the Hst54-15-Spd conjugates by performing atomistic molecular dynamics simulations in combination with small-angle X-ray scattering. It was shown that the Hst54-15-Spd conjugates adopt extended and slightly rigid random coil conformations without any secondary structure in aqueous solution. It is hypothesized that the increased fungal killing potential of Hst54-15-Spd, in comparison with the Spd-Hst54-15 conjugate, is due to the more extended conformations of the former, which cause the bonded Spd molecule to be more accessible for recognition by polyamine transporters in the cell

Histatin 5 resistance of Candida glabrata can be reversed by insertion of Candida albicans polyamine transporter-encoding genes DUR3 and DUR31.

Candida albicans and Candida glabrata are predominant fungi associated with oral candidiasis. Histatin 5 (Hst 5) is a small cationic human salivary peptide with high fungicidal activity against C. albicans, however many strains of C. glabrata are resistant. Since Hst 5 requires fungal binding to cell wall components prior to intracellular translocation, reduced Hst 5 binding to C. glabrata may be the reason for its insensitivity. C. glabrata has higher surface levels of β-1,3-glucans as compared with C. albicans; however these differences did not account for reduced Hst 5 uptake and killing in C. glabrata. Similarly, the biofilm matrix of C. glabrata contained significantly higher levels of β-1,3-glucans compared with C. albicans, but it did not reduce the percentage of Hst 5 positive fungal cells in the biofilm. Hst 5 enters C. albicans cell through polyamine transporters Dur3p and Dur31p that are uncharacterized in C. glabrata. C. glabrata strains expressing CaDur3 and CaDur31 had two-fold higher killing and uptake of Hst 5. Thus, neither C. glabrata cell surface or biofilm matrix β-1,3-glucan levels affected Hst 5 toxicity; rather the crucial rate limiting step is reduced uptake that can be overcome by expression of C. albicans Dur proteins in C. glabrata

Salivary metals, age, and gender correlate with cultivable oral Candida carriage levels

Background: Little is known about the normal range of metal levels in unstimulated saliva, nor whether these might impact Candida carriage in healthy individuals. Both are important in determining which populations are at risk for candidiasis, as the availability of metal ions can influence the growth and pathogenesis of Candida albicans. Objective: We quantified salivary metals of healthy individuals to determine the correlation with C. albicans oral colonization. Design: Unstimulated whole saliva was collected from healthy adults and plated to determine fungal carriage, and metal content was measured using ICP-mass spectrometry (ICP-MS). Results: Zinc was most abundant, followed by iron, copper, manganese, and nickel. Cultivable oral Candida carriage was found in 48% of people. Total protein levels did not differ in salivas from donors with or without carriage. However, innate fungicidal activity was increased in donors with carriage; correlations between levels of several metals were stronger in salivas with fungal carriage, indicating a shift in the oral environment. Concentrations of copper and manganese, as well as age and gender, were significantly predictive of carriage levels in a multiple regression model. Conclusions: Salivary copper and manganese content along with age and gender could be used as a predictive metric for individuals that are more susceptible to Candida overgrowth

Candida albicans Shed Msb2 and Host Mucins Affect the Candidacidal Activity of Salivary Hst 5

Salivary Histatin 5 (Hst 5) is an antimicrobial peptide that exhibits potent antifungal activity towards Candida albicans, the causative agent of oral candidiasis. However, it exhibits limited activity in vivo, largely due to inactivation by salivary components of both host and pathogen origin. Proteins secreted by C. albicans during infection such as secreted aspartyl proteases (Saps) and shed mucin Msb2 can reduce Hst 5 activity; and human salivary mucins, while suggested to protect Hst 5 from proteolytic degradation, can entrap peptides into mucin gels, thereby reducing bioavailability. We show here that Sap6 that is secreted during hyphal growth reduces Hst 5 activity, most likely a result of proteolytic degradation of Hst 5 since this effect is abrogated with heat inactivated Sap 6. We further show that just like C. albicans shedding Msb2, mammalian mucins, fetuin and porcine gut mucin (that is related to salivary mucins), also reduce Hst 5 activity. However, we identify mucin-like protein-induced changes in C. albicans cell morphology and aggregation patterns, suggesting that the effect of such proteins on Hst 5 cannot be interpreted independently of their effect on yeast cells

Candida albicans Shed Msb2 and Host Mucins Affect the Candidacidal Activity of Salivary Hst 5

Salivary Histatin 5 (Hst 5) is an antimicrobial peptide that exhibits potent antifungal activity towards Candida albicans, the causative agent of oral candidiasis. However, it exhibits limited activity in vivo, largely due to inactivation by salivary components of both host and pathogen origin. Proteins secreted by C. albicans during infection such as secreted aspartyl proteases (Saps) and shed mucin Msb2 can reduce Hst 5 activity; and human salivary mucins, while suggested to protect Hst 5 from proteolytic degradation, can entrap peptides into mucin gels, thereby reducing bioavailability. We show here that Sap6 that is secreted during hyphal growth reduces Hst 5 activity, most likely a result of proteolytic degradation of Hst 5 since this effect is abrogated with heat inactivated Sap 6. We further show that just like C. albicans shedding Msb2, mammalian mucins, fetuin and porcine gut mucin (that is related to salivary mucins), also reduce Hst 5 activity. However, we identify mucin-like protein-induced changes in C. albicans cell morphology and aggregation patterns, suggesting that the effect of such proteins on Hst 5 cannot be interpreted independently of their effect on yeast cells

Secreted aspartic protease cleavage of Candida albicans Msb2 activates Cek1 MAPK signaling affecting biofilm formation and oropharyngeal candidiasis.

Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence