Luiz R. Nunes

Comparative Genomics of Two Leptospira interrogans Serovars Reveals Novel Insights into Physiology and Pathogenesis

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organisms complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Comparative Analyses of the Complete Genome Sequences of Pierce's Disease and Citrus Variegated Chlorosis Strains of Xylella fastidiosa

Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X. fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierces disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X. fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X. fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X. fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies.

Expressed sequence tag analysis of the human pathogen Paracoccidioides brasiliensis yeast phase: identification of putative homologues of Candida albicans virulence and pathogenicity genes.

ABSTRACT Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis. We present here a survey of expressed genes in the yeast pathogenic phase of P. brasiliensis. We obtained 13,490 expressed sequence tags from both 5′ and 3′ ends. Clustering analysis yielded the partial sequences of 4,692 expressed genes that were functionally classified by similarity to known genes. We have identified several Candida albicans virulence and pathogenicity homologues in P. brasiliensis. Furthermore, we have analyzed the expression of some of these genes during the dimorphic yeast-mycelium-yeast transition by real-time quantitative reverse transcription-PCR. Clustering analysis of the mycelium-yeast transition revealed three groups: (i) RBT, hydrophobin, and isocitrate lyase; (ii) malate dehydrogenase, contigs Pb1067 and Pb1145, GPI, and alternative oxidase; and (iii) ubiquitin, delta-9-desaturase, HSP70, HSP82, and HSP104. The first two groups displayed high mRNA expression in the mycelial phase, whereas the third group showed higher mRNA expression in the yeast phase. Our results suggest the possible conservation of pathogenicity and virulence mechanisms among fungi, expand considerably gene identification in P. brasiliensis, and provide a broader basis for further progress in understanding its biological peculiarities.

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli.

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.

Transcriptome Analysis of Paracoccidioides brasiliensis Cells Undergoing Mycelium-to-Yeast Transition

ABSTRACT Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), a systemic mycosis prevalent in South America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and transform into the yeast parasitic form. Thus, the mycelium-to-yeast transition is of particular interest because conversion to yeast is essential for infection. We have used a P. brasiliensis biochip carrying sequences of 4,692 genes from this fungus to monitor gene expression at several time points of the mycelium-to-yeast morphological shift (from 5 to 120 h). The results revealed a total of 2,583 genes that displayed statistically significant modulation in at least one experimental time point. Among the identified gene homologues, some encoded enzymes involved in amino acid catabolism, signal transduction, protein synthesis, cell wall metabolism, genome structure, oxidative stress response, growth control, and development. The expression pattern of 20 genes was independently verified by real-time reverse transcription-PCR, revealing a high degree of correlation between the data obtained with the two methodologies. One gene, encoding 4-hydroxyl-phenyl pyruvate dioxygenase (4-HPPD), was highly overexpressed during the mycelium-to-yeast differentiation, and the use of NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione], a specific inhibitor of 4-HPPD activity, as well as that of NTBC derivatives, was able to inhibit growth and differentiation of the pathogenic yeast phase of the fungus in vitro. These data set the stage for further studies involving NTBC and its derivatives as new chemotherapeutic agents against PCM and confirm the potential of array-based approaches to identify new targets for the development of alternative treatments against pathogenic microorganisms.

Analysis of gene expression in two growth states of Xylella fastidiosa and its relationship with pathogenicity

Xylella fastidiosa is a plant pathogen responsible for diseases of economically important crops. Although there is considerable disagreement about its mechanism of pathogenicity, blockage of the vessels is one of the most accepted hypotheses. Loss of virulence by this bacterium was observed after serial passages in axenic culture. To confirm the loss of pathogenicity of X. fastidiosa, the causing agent of citrus variegated chlorosis (CVC), freshly-isolated bacteria (first passage [FP] condition) as well as bacteria obtained after 46 passages in axenic culture (several passage [SP] condition) were inoculated into sweet orange and periwinkle plants. Using real time quantitative polymerase chain reaction, we verified that the colonization of FP cells was more efficient for both hosts. The sequence of the complete X. fastidiosa genome allowed the construction of a DNA microarray that was used to investigate the total changes in gene expression associated with the FP condition. Most genes found to be induced in the FP condition were associated with adhesion and probably with adaptation to the host environment. This report represents the first study of the transcriptome of this pathogen, which has recently gained more importance, since the genome of several strains has been either partially or entirely sequenced.

Evaluation of Monocot and Eudicot Divergence Using the Sugarcane Transcriptome

Over 40,000 sugarcane (Saccharum officinarum) consensus sequences assembled from 237,954 expressed sequence tags were compared with the protein and DNA sequences from other angiosperms, including the genomes of Arabidopsis and rice (Oryza sativa). Approximately two-thirds of the sugarcane transcriptome have similar sequences in Arabidopsis. These sequences may represent a core set of proteins or protein domains that are conserved among monocots and eudicots and probably encode for essential angiosperm functions. The remaining sequences represent putative monocot-specific genetic material, one-half of which were found only in sugarcane. These monocot-specific cDNAs represent either novelties or, in many cases, fast-evolving sequences that diverged substantially from their eudicot homologs. The wide comparative genome analysis presented here provides information on the evolutionary changes that underlie the divergence of monocots and eudicots. Our comparative analysis also led to the identification of several not yet annotated putative genes and possible gene loss events in Arabidopsis.

Transcriptome analysis and molecular studies on sulfur metabolism in the human pathogenic fungus Paracoccidioides brasiliensis

The dimorphic pathogenic fungus Paracoccidioides brasiliensis can grow as a prototroph for organic sulfur as a mycelial (non-pathogenic) form, but it is unable to assimilate inorganic sulfur as a yeast (pathogenic) form. Temperature and the inability to assimilate inorganic sulfur are the single conditions known to affect P. brasiliensis mycelium-to-yeast (M–Y) dimorphic transition. For a comprehensive evaluation of genes that have their expression modulated during the M–Y transition in different culture media, we performed a large-scale analysis of gene expression using a microarray hybridization approach. The results of the present work demonstrate the use of microarray hybridization analysis to examine gene expression during the M–Y transition in minimal medium and compare these results with the M–Y transition in complete medium. Our results showed that about 95% of the genes in our microarray are mainly responding to the temperature trigger, independently of the media where the M–Y transition took place. As a preliminary step to understand the inorganic sulfur inability in P. brasiliensis yeast form, we decided to characterize the mRNA accumulation of several genes involved in different aspects of both organic and inorganic sulfur assimilation. Our results suggest that although P. brasiliensis cannot use inorganic sulfur as a single sulfur source to initiate both M–Y transition and Y growth, the fungus can somehow use both organic and inorganic pathways during these growth processes.

Comparative genomic characterization of citrus-associated Xylella fastidiosa strains.

BackgroundThe xylem-inhabiting bacterium Xylella fastidiosa (Xf) is the causal agent of Pierces disease (PD) in vineyards and citrus variegated chlorosis (CVC) in orange trees. Both of these economically-devastating diseases are caused by distinct strains of this complex group of microorganisms, which has motivated researchers to conduct extensive genomic sequencing projects with Xf strains. This sequence information, along with other molecular tools, have been used to estimate the evolutionary history of the group and provide clues to understand the capacity of Xf to infect different hosts, causing a variety of symptoms. Nonetheless, although significant amounts of information have been generated from Xf strains, a large proportion of these efforts has concentrated on the study of North American strains, limiting our understanding about the genomic composition of South American strains – which is particularly important for CVC-associated strains.ResultsThis paper describes the first genome-wide comparison among South American Xf strains, involving 6 distinct citrus-associated bacteria. Comparative analyses performed through a microarray-based approach allowed identification and characterization of large mobile genetic elements that seem to be exclusive to South American strains. Moreover, a large-scale sequencing effort, based on Suppressive Subtraction Hybridization (SSH), identified 290 new ORFs, distributed in 135 Groups of Orthologous Elements, throughout the genomes of these bacteria.ConclusionResults from microarray-based comparisons provide further evidence concerning activity of horizontally transferred elements, reinforcing their importance as major mediators in the evolution of Xf. Moreover, the microarray-based genomic profiles showed similarity between Xf strains 9a5c and Fb7, which is unexpected, given the geographical and chronological differences associated with the isolation of these microorganisms. The newly identified ORFs, obtained by SSH, represent an approximately 10% increase in our current knowledge of the South American Xf gene pool and include new putative virulence factors, as well as novel potential markers for strain identification. Surprisingly, this list of novel elements include sequences previously believed to be unique to North American strains, pointing to the necessity of revising the list of specific markers that may be used for identification of distinct Xf strains.

Transcriptome Analysis of the Phytobacterium Xylella fastidiosa Growing under Xylem-Based Chemical Conditions

Xylella fastidiosa is a xylem-limited bacterium responsible for important plant diseases, like citrus-variegated chlorosis (CVC) and grapevine Pierces disease (PD). Interestingly, in vitro growth of X. fastidiosa in chemically defined media that resemble xylem fluid has been achieved, allowing studies of metabolic processes used by xylem-dwelling bacteria to thrive in such nutrient-poor conditions. Thus, we performed microarray hybridizations to compare transcriptomes of X. fastidiosa cells grown in 3G10-R, a medium that resembles grape sap, and in Periwinkle Wilt (PW), the complex medium traditionally used to cultivate X. fastidiosa. We identified 299 transcripts modulated in response to growth in these media. Some 3G10R-overexpressed genes have been shown to be upregulated in cells directly isolated from infected plants and may be involved in plant colonization, virulence and environmental competition. In contrast, cells cultivated in PW show a metabolic switch associated with increased aerobic respiration and enhanced bacterial growth rates.