The Small RNA Teg41 Is a Pleiotropic Regulator of Virulence in Staphylococcus aureus.

Previously, our group demonstrated a role for the small RNA (sRNA) Teg41 in regulating production of the alpha phenol-soluble modulin toxins (αPSMs) in Staphylococcus aureus. Overexpressing Teg41 increased αPSM production while deleting the 3' end of Teg41 (Teg41Δ3' strain) resulted in a decrease in αPSM production, reduced hemolytic activity of S. aureus culture supernatants, and attenuated virulence in a murine abscess model of infection. In this study, we further explore the attenuation of virulence in the Teg41Δ3' strain. Using both localized and systemic models of infection, we demonstrate that the Teg41Δ3' strain is more severely attenuated than an ΔαPSM mutant, strongly suggesting that Teg41 influences more than the αPSMs. Proteomic and transcriptomic analysis of the wild-type and Teg41Δ3' strains reveals widespread alterations in transcript abundance and protein production in the absence of Teg41, confirming that Teg41 has pleiotropic effects in the cell. We go on to investigate the molecular mechanism underlying Teg41-mediated gene regulation. Surprisingly, results demonstrate that certain Teg41 target genes, including the αPSMs and ßPSMs, are transcriptionally altered in the Teg41Δ3' strain, while other targets, specifically spa (encoding surface protein A), are regulated at the level of transcript stability. Collectively, these data demonstrate that Teg41 is a pleiotropic RNA regulator in S. aureus that influences expression of a variety of genes using multiple different mechanisms.

The novel protein ScrA acts through the SaeRS two-component system to regulate virulence gene expression in Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive commensal that can also cause a variety of infections in humans. S. aureus virulence factor gene expression is under tight control by a complex regulatory network, which includes, sigma factors, sRNAs, and two-component systems (TCS). Previous work in our laboratory demonstrated that overexpression of the sRNA tsr37 leads to an increase in bacterial aggregation. Here, we demonstrate that the clumping phenotype is dependent on a previously unannotated 88 amino acid protein encoded within the tsr37 sRNA transcript (which we named ScrA for S. aureus clumping regulator A). To investigate the mechanism of action of ScrA we performed proteomics and transcriptomics in a ScrA overexpressing strain and show that a number of surface adhesins are upregulated, while secreted proteases are downregulated. Results also showed upregulation of the SaeRS TCS, suggesting that ScrA is influencing SaeRS activity. Overexpression of ScrA in a saeR mutant abrogates the clumping phenotype confirming that ScrA functions via the Sae system. Finally, we identified the ArlRS TCS as a positive regulator of scrA expression. Collectively, our results show that ScrA is an activator of the SaeRS system and suggests that ScrA may act as an intermediary between the ArlRS and SaeRS systems.

Temperature Influences the Composition and Cytotoxicity of Extracellular Vesicles in Staphylococcus aureus.

Staphylococcus aureus is a pathogenic bacterium but also a commensal of skin and anterior nares in humans. As S. aureus transits from skins/nares to inside the human body, it experiences changes in temperature. The production and content of S. aureus extracellular vesicles (EVs) have been increasingly studied over the past few years, and EVs are increasingly being recognized as important to the infectious process. Nonetheless, the impact of temperature variation on S. aureus EVs has not been studied in detail, as most reports that investigate EV cargoes and host cell interactions are performed using vesicles produced at 37°C. Here, we report that EVs in S. aureus differ in size and protein/RNA cargo depending on the growth temperature used. We demonstrate that the temperature-dependent regulation of vesicle production in S. aureus is mediated by the alpha phenol-soluble modulin peptides (αPSMs). Through proteomic analysis, we observed increased packaging of virulence factors at 40°C, whereas the EV proteome has greater diversity at 34°C. Similar to the protein content, we perform transcriptomic analysis and demonstrate that the RNA cargo also is impacted by temperature. Finally, we demonstrate greater αPSM- and alpha-toxin-mediated erythrocyte lysis with 40°C EVs, but 34°C EVs are more cytotoxic toward THP-1 cells. Together, our study demonstrates that small temperature variations have great impact on EV biogenesis and shape the interaction with host cells. IMPORTANCE Extracellular vesicles (EVs) are lipid bilayer spheres that contain proteins, nucleic acids, and lipids secreted by bacteria. They are involved in Staphylococcus aureus infections, as they package virulence factors and deliver their contents inside host cells. The impact of temperature variations experienced by S. aureus during the infectious process on EVs is unknown. Here, we demonstrate the importance of temperature in vesicle production and packaging. High temperatures promote packaging of virulence factors and increase the protein and lipid concentration but reduce the overall RNA abundance and protein diversity in EVs. The importance of temperature changes is highlighted by the fact that EVs produced at low temperature are more toxic toward macrophages, whereas EVs produced at high temperature display more hemolysis toward erythrocytes. Our research brings new insights into temperature-dependent vesiculation and interaction with the host during S. aureus transition from colonization to virulence.

Detection and Quantification of Secreted Nuclease Activity in Staphylococcus aureus Culture Supernatants.

Staphylococcal secreted nuclease contributes to S. aureus virulence by degrading neutrophil extracellular traps (NETs), which allows the bacterium to evade the host immune system and has also been shown to promote biofilm dispersal. In this chapter, two methods for detecting nuclease activity are described, both of which have increased sensitivity compared to the traditional nuclease agar method.

Staphylococcus aureus Responds to Physiologically Relevant Temperature Changes by Altering Its Global Transcript and Protein Profile.

Staphylococcus aureus is an opportunistic pathogen that colonizes the anterior nares of 30 to 50% of the population. Colonization is most often asymptomatic; however, self-inoculation can give rise to potentially fatal infections of the deeper tissues and blood. Like all bacteria, S. aureus can sense and respond to environmental cues and modify gene expression to adapt to specific environmental conditions. The transition of S. aureus from the nares to the deeper tissues and blood is accompanied by changes in environmental conditions, such as nutrient availability, pH, and temperature. In this study, we perform transcriptomics and proteomics on S. aureus cultures growing at three physiologically relevant temperatures, 34°C (nares), 37°C (body), and 40°C (pyrexia), to determine if small scale, biologically meaningful alterations in temperature impact S. aureus gene expression. Results show that small but definite temperature changes elicit a large-scale restructuring of the S. aureus transcriptome and proteome in a manner that, most often, inversely correlates with increasing temperature. We also provide evidence that a large majority of these changes are modulated at the posttranscriptional level, possibly by sRNA regulatory elements. Phenotypic analyses were also performed to demonstrate that these changes have physiological relevance. Finally, we investigate the impact of temperature-dependent alterations in gene expression on S. aureus pathogenesis and demonstrate decreased intracellular invasion of S. aureus grown at 34°C. Collectively, our results demonstrate that small but biologically meaningful alterations in temperature influence S. aureus gene expression, a process that is likely a major contributor to the transition from a commensal to pathogen.IMPORTANCE Enteric bacterial pathogens, like Escherichia coli, are known to experience large temperature differences as they are transmitted through the fecal oral route. This change in temperature has been demonstrated to influence bacterial gene expression and facilitate infection. Staphylococcus aureus is a human-associated pathogen that can live as a commensal on the skin and nares or cause invasive infections of the deeper tissues and blood. Factors influencing S. aureus nasal colonization are not fully understood; however, individuals colonized with S. aureus are at increased risk of invasive infections through self-inoculation. The transition of S. aureus from the nose (colonization) to the body (infection) is accompanied by a modest but definite temperature increase, from 34°C to 37°C. In this study, we investigate whether these host-associated small temperature changes can influence S. aureus gene expression. Results show widespread changes in the bacterial transcriptome and proteome at three physiologically relevant temperatures (34°C, 37°C, and 40°C).

Staphylococcus aureus Trigger Factor Is Involved in Biofilm Formation and Cooperates with the Chaperone PpiB.

Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that assist in protein folding around proline-peptide bonds, and they often possess chaperone activity. Staphylococcus aureus encodes three PPIases, i.e., PrsA, PpiB, and trigger factor (TF). Previous work by our group demonstrated a role for both PrsA and PpiB in S. aureus; however, TF remains largely unstudied. Here, we identify a role for TF in S. aureus biofilm formation and demonstrate cooperation between TF and the cytoplasmic PPIase PpiB. Mutation of the tig gene (encoding TF) led to reduced biofilm development in vitro but no significant attenuation of virulence in a mouse model of infection. To investigate whether TF possesses chaperone activity, we analyzed the ability of a tig mutant to survive acid and base stress. While there was no significant decrease for a tig mutant, a ppiBtig double mutant exhibited significant decreases in cell viability after acid and base challenges. We then demonstrated that a ppiB tig double mutant had exacerbated phenotypes in vitro and in vivo, compared to either single mutant. Finally, in vivo immunoprecipitation of epitope-tagged PpiB revealed that PpiB interacted with 4 times the number of proteins when TF was absent from the cell, suggesting that it may be compensating for the loss of TF. Interestingly, the only proteins found to interact with TF were TF itself, fibronectin-binding protein B (FnBPB), and the chaperone protein ClpB. Collectively, these results support the first phenotype for S. aureus TF and demonstrate a greater network of cooperation between chaperone proteins in Staphylococcus aureusIMPORTANCES. aureus encodes a large number of virulence factors that aid the bacterium in survival and pathogenesis. These virulence factors have a wide variety of functions; however, they must all be properly secreted in order to be functional. Bacterial chaperone proteins often assist in secretion by trafficking proteins to secretion machinery or assisting in proper protein folding. Here, we report that the S. aureus chaperone TF contributes to biofilm formation and cooperates with the chaperone PpiB to regulate S. aureus virulence processes. These data highlight the first known role for TF in S. aureus and suggest that S. aureus chaperone proteins may be involved in a greater regulatory network in the cell.

Reading between the Lines: Utilizing RNA-Seq Data for Global Analysis of sRNAs in Staphylococcus aureus.

Regulatory small RNAs (sRNAs) are known to play important roles in the Gram-positive bacterial pathogen Staphylococcus aureus; however, their existence is often overlooked, primarily because sRNA genes are absent from genome annotation files. Consequently, transcriptome sequencing (RNA-Seq)-based experimental approaches, performed using standard genome annotation files as a reference, have likely overlooked data for sRNAs. Previously, we created an updated S. aureus genome annotation file, which included annotations for 303 known sRNAs in USA300. Here, we utilized this updated reference file to reexamine publicly available RNA-Seq data sets in an attempt to recover lost information on sRNA expression, stability, and potential to encode peptides. First, we used transcriptomic data from 22 studies to identify how the expression of 303 sRNAs changed under 64 different experimental conditions. Next, we used RNA-Seq data from an RNA stability assay to identify highly stable/unstable sRNAs. We went on to reanalyze a ribosome profiling (Ribo-seq) data set to identify sRNAs that have the potential to encode peptides and to experimentally confirm the presence of three of these peptides in the USA300 background. Interestingly, one of these sRNAs/peptides, encoded at the tsr37 locus, influences the ability of S. aureus cells to autoaggregate. Finally, we reexamined two recently published in vivo RNA-Seq data sets, from the cystic fibrosis (CF) lung and a murine vaginal colonization study, and identified 29 sRNAs that may play a role in vivo Collectively, these results can help inform future studies of these important regulatory elements in S. aureus and highlight the need for ongoing curating and updating of genome annotation files.IMPORTANCE Regulatory small RNAs (sRNAs) are a class of RNA molecules that are produced in bacterial cells but that typically do not encode proteins. Instead, they perform a variety of critical functions within the cell as RNA. Most bacterial genomes do not include annotations for sRNA genes, and any type of analysis that is performed using a bacterial genome as a reference will therefore overlook data for sRNAs. In this study, we reexamined hundreds of previously generated S. aureus RNA-Seq data sets and reanalyzed them to generate data for sRNAs. To do so, we utilized an updated S. aureus genome annotation file, previously generated by our group, which contains annotations for 303 sRNAs. The data generated (which were previously discarded) shed new light on sRNAs in S. aureus, most of which are unstudied, and highlight certain sRNAs that are likely to play important roles in the cell.

Novel Regulation of Alpha-Toxin and the Phenol-Soluble Modulins by Peptidyl-Prolyl cis/trans Isomerase Enzymes in Staphylococcus aureus.

Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that catalyze the cis-to-trans isomerization around proline bonds, allowing proteins to fold into their correct confirmation. Previously, we identified two PPIase enzymes in Staphylococcus aureus (PpiB and PrsA) that are involved in the regulation of virulence determinants and have shown that PpiB contributes to S. aureus virulence in a murine abscess model of infection. Here, we further examine the role of these PPIases in S. aureus virulence and, in particular, their regulation of hemolytic toxins. Using murine abscess and systemic models of infection, we show that a ppiB mutant in a USA300 background is attenuated for virulence but that a prsA mutant is not. Deletion of the ppiB gene leads to decreased bacterial survival in macrophages and nasal epithelial cells, while there is no significant difference when prsA is deleted. Analysis of culture supernatants reveals that a ppiB mutant strain has reduced levels of the phenol-soluble modulins and that both ppiB and prsA mutants have reduced alpha-toxin activity. Finally, we perform immunoprecipitation to identify cellular targets of PpiB and PrsA. Results suggest a novel role for PpiB in S. aureus protein secretion. Collectively, our results demonstrate that PpiB and PrsA influence S. aureus toxins via distinct mechanisms, and that PpiB but not PrsA contributes to disease.

An unconventional RNA-based thermosensor within the 5' UTR of Staphylococcus aureus cidA.

Staphylococcus aureus is a Gram-positive bacterial pathogen of global concern and a leading cause of bacterial infections worldwide. Asymptomatic carriage of S. aureus on the skin and in the anterior nares is common and recognized as a predisposing factor to invasive infection. Transition of S. aureus from the carriage state to that of invasive infection is often accompanied by a temperature upshift from approximately 33°C to 37°C. Such a temperature shift is known in other pathogens to influence gene expression, often resulting in increased production of factors that promote survival or virulence within the host. One mechanism by which bacteria modulate gene expression in response to temperature is by the regulatory activity of RNA-based thermosensors, cis-acting riboregulators that control translation efficiency. This study was designed to identify and characterize RNA-based thermosensors in S. aureus. Initially predicted by in silico analyses of the S. aureus USA300 genome, reporter-based gene expression analyses and site-specific mutagenesis were performed to demonstrate the presence of a functional thermosensor within the 5' UTR of cidA, a gene implicated in biofilm formation and survival of the pathogen. The nucleic sequence composing the identified thermosensor are sufficient to confer temperature-dependent post-transcriptional regulation, and activity is predictably altered by the introduction of site-specific mutations designed to stabilize or destabilize the structure within the identified thermosensor. The identified regulator is functional in both the native bacterial host S. aureus and in the distally related species Escherichia coli, suggesting that its regulatory activity is independent of host-specific factors. Interestingly, unlike the majority of bacterial RNA-based thermosensors characterized to date, the cidA thermosensor facilitates increased target gene expression at lower temperatures. In addition to the characterization of the first RNA-based thermosensor in the significant pathogen S. aureus, it highlights the diversity of function within this important class of ribo-regulators.

The Small RNA Teg41 Regulates Expression of the Alpha Phenol-Soluble Modulins and Is Required for Virulence in Staphylococcus aureus.

Small RNAs (sRNAs) remain an understudied class of regulatory molecules in bacteria in general and in Gram-positive bacteria in particular. In the major human pathogen Staphylococcus aureus, hundreds of sRNAs have been identified; however, only a few have been characterized in detail. In this study, we investigate the role of the sRNA Teg41 in S. aureus virulence. We demonstrate that Teg41, an sRNA divergently transcribed from the locus that encodes the cytolytic alpha phenol-soluble modulin (αPSM) peptides, plays a critical role in αPSM production. Overproduction of Teg41 leads to an increase in αPSM levels and a corresponding increase in hemolytic activity from S. aureus cells and cell-free culture supernatants. To identify regions of Teg41 important for its function, we performed an in silico RNA-RNA interaction analysis which predicted an interaction between the 3' end of Teg41 and the αPSM transcript. Deleting a 24-nucleotide region from the S. aureus genome, corresponding to the 3' end of Teg41, led to a 10-fold reduction in αPSM-dependent hemolytic activity and attenuation of virulence in a murine abscess model of infection. Restoration of hemolytic activity in the Teg41Δ3' strain was possible by expressing full-length Teg41 in trans Restoration of hemolytic activity was also possible by expressing the 3' end of Teg41, suggesting that this region of Teg41 is necessary and sufficient for αPSM-dependent hemolysis. Our results show that Teg41 is positively influencing αPSM production, demonstrating for the first time regulation of the αPSM peptides by an sRNA in S. aureusIMPORTANCE The alpha phenol-soluble modulins (αPSMs) are among the most potent toxins produced by Staphylococcus aureus Their biological role during infection has been studied in detail; however, the way they are produced by the bacterial cell is not well understood. In this work, we identify a small RNA molecule called Teg41 that plays an important role in αPSM production by S. aureus Teg41 positively influences αPSM production. The importance of Teg41 is highlighted by the fact that a strain containing a deletion in the 3' end of Teg41 produces significantly less αPSMs and is attenuated for virulence in a mouse abscess model of infection. As the search for new therapeutic strategies to combat S. aureus infection proceeds, Teg41 may represent a novel target.

The Intracellular Cyclophilin PpiB Contributes to the Virulence of Staphylococcus aureus Independently of Its Peptidyl-Prolyl cis/trans Isomerase Activity.

The Staphylococcus aureus cyclophilin PpiB is an intracellular peptidyl prolyl cis/trans isomerase (PPIase) that has previously been shown to contribute to secreted nuclease and hemolytic activity. In this study, we investigated the contribution of PpiB to S. aureus virulence. Using a murine abscess model of infection, we demonstrated that a ppiB mutant is attenuated for virulence. We went on to investigate the mechanism through which PpiB protein contributes to virulence, in particular the contribution of PpiB PPIase activity. We determined the amino acid residues that are important for PpiB PPIase activity and showed that a single amino acid substitution (F64A) completely abrogates PPIase activity. Using purified PpiB F64A protein in vitro, we showed that PPIase activity only partially contributes to Nuc refolding and that PpiB also possesses PPIase-independent activity. Using allelic exchange, we introduced the F64A substitution onto the S. aureus chromosome, generating a strain that produces enzymatically inactive PpiB. Analysis of the PpiB F64A strain revealed that PPIase activity is not required for hemolysis of human blood or virulence in a mouse. Together, these results demonstrate that PpiB contributes to S. aureus virulence via a mechanism unrelated to prolyl isomerase activity.

An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease.

Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease.

Composition, Assembly, and Trafficking of a Wheat Xylan Synthase Complex.

Xylans play an important role in plant cell wall integrity and have many industrial applications. Characterization of xylan synthase (XS) complexes responsible for the synthesis of these polymers is currently lacking. We recently purified XS activity from etiolated wheat (Triticum aestivum) seedlings. To further characterize this purified activity, we analyzed its protein composition and assembly. Proteomic analysis identified six main proteins: two glycosyltransferases (GTs) TaGT43-4 and TaGT47-13; two putative mutases (TaGT75-3 and TaGT75-4) and two non-GTs; a germin-like protein (TaGLP); and a vernalization related protein (TaVER2). Coexpression of TaGT43-4, TaGT47-13, TaGT75-3, and TaGT75-4 in Pichia pastoris confirmed that these proteins form a complex. Confocal microscopy showed that all these proteins interact in the endoplasmic reticulum (ER) but the complexes accumulate in Golgi, and TaGT43-4 acts as a scaffold protein that holds the other proteins. Furthermore, ER export of the complexes is dependent of the interaction between TaGT43-4 and TaGT47-13. Immunogold electron microscopy data support the conclusion that complex assembly occurs at specific areas of the ER before export to the Golgi. A di-Arg motif and a long sequence motif within the transmembrane domains were found conserved at the NH2-terminal ends of TaGT43-4 and homologous proteins from diverse taxa. These conserved motifs may control the forward trafficking of the complexes and their accumulation in the Golgi. Our findings indicate that xylan synthesis in grasses may involve a new regulatory mechanism linking complex assembly with forward trafficking and provide new insights that advance our understanding of xylan biosynthesis and regulation in plants.

Genome-wide Annotation, Identification, and Global Transcriptomic Analysis of Regulatory or Small RNA Gene Expression in Staphylococcus aureus.

UNLABELLED: In Staphylococcus aureus, hundreds of small regulatory or small RNAs (sRNAs) have been identified, yet this class of molecule remains poorly understood and severely understudied. sRNA genes are typically absent from genome annotation files, and as a consequence, their existence is often overlooked, particularly in global transcriptomic studies. To facilitate improved detection and analysis of sRNAs in S. aureus, we generated updated GenBank files for three commonly used S. aureus strains (MRSA252, NCTC 8325, and USA300), in which we added annotations for >260 previously identified sRNAs. These files, the first to include genome-wide annotation of sRNAs in S. aureus, were then used as a foundation to identify novel sRNAs in the community-associated methicillin-resistant strain USA300. This analysis led to the discovery of 39 previously unidentified sRNAs. Investigating the genomic loci of the newly identified sRNAs revealed a surprising degree of inconsistency in genome annotation in S. aureus, which may be hindering the analysis and functional exploration of these elements. Finally, using our newly created annotation files as a reference, we perform a global analysis of sRNA gene expression in S. aureus and demonstrate that the newly identified tsr25 is the most highly upregulated sRNA in human serum. This study provides an invaluable resource to the S. aureus research community in the form of our newly generated annotation files, while at the same time presenting the first examination of differential sRNA expression in pathophysiologically relevant conditions. IMPORTANCE: Despite a large number of studies identifying regulatory or small RNA (sRNA) genes in Staphylococcus aureus, their annotation is notably lacking in available genome files. In addition to this, there has been a considerable lack of cross-referencing in the wealth of studies identifying these elements, often leading to the same sRNA being identified multiple times and bearing multiple names. In this work, we have consolidated and curated known sRNA genes from the literature and mapped them to their position on the S. aureus genome, creating new genome annotation files. These files can now be used by the scientific community at large in experiments to search for previously undiscovered sRNA genes and to monitor sRNA gene expression by transcriptome sequencing (RNA-seq). We demonstrate this application, identifying 39 new sRNAs and studying their expression during S. aureus growth in human serum.

α-Fucosidases with different substrate specificities from two species of Fusarium.

Two fungal-secreted α-fucosidases and their genes were characterized. FoFCO1 was purified from culture filtrates of Fusarium oxysporum strain 0685 grown on L-fucose and its encoding gene identified in the sequenced genome of strain 4287. FoFCO1 was active on p-nitrophenyl-α-fucoside (pNP-Fuc), but did not defucosylate a nonasaccharide (XXFG) fragment of pea xyloglucan. A putative α-fucosidase gene (FgFCO1) from Fusarium graminearum was expressed in Pichia pastoris. FgFCO1 was ~1,800 times less active on pNP-Fuc than FoFCO1, but was able to defucosylate the XXFG nonasaccharide. Although FgFCO1 and FoFCO1 both belong to Glycosyl Hydrolase family 29, they share <25 % overall amino acid identity. Alignment of all available fungal orthologs of FoFCO1 and FgFCO1 indicated that these two proteins belong to two subfamilies of fungal GH29 α-fucosidases. Fungal orthologs of subfamily 1 (to which FoFCO1 belongs) are taxonomically more widely distributed than subfamily 2 (FgFCO1), but neither was universally present in the sequenced fungal genomes. Trichoderma reesei and most species of Aspergillus lack genes for either GH29 subfamily.