extract the genes from the following content. the gene is names as  gudiv_xxx where xxx is number. also you need to classify the genes by surface molecules (variable surface lipoproteins and membrane nuclease lipoproteins), urease, phospholipase, hemolysin, MIB-MIP system, and capsule. 
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Virulence and pathogenicity mechanisms

To date, only few features have been associated with virulence of U. diversum, such as the activity of urease and phospholipase, and its ability to invade some eukaryotic cells [8]. After analyzing the genome of U. diversum, it was possible to find CDSs that are likely related to bacterial pathogenicity and can expand our knowledge regarding the mechanisms by which U. diversum causes disease. CDSs of urease (gudiv_255, gudiv_254, gudiv_253, gudiv_252, gudiv_251, gudiv_250, and gudiv_249), phospholipase (gudiv_472), hemolysin (gudiv_91), and a Mycoplasma Ig binding protein (MIB)—Mycoplasma Ig protease (MIP) system (gudiv_161/gudiv_162 and gudiv_612/gudiv_613) were found and are described below in more detail. In addition, CDSs that are likely related to the production of a capsular structure (gudiv_216) and surface molecules were observed. A scheme of these virulence factors is found in Fig 4A.
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Fig 4. Virulence and pathogenicity mechanisms.

(A) Virulence map of U. diversum ATCC 49782. (B) Schematic representation of the urease gene cluster from U. diversum ATCC 49782. Structural subunits: ureA (gudiv_255), ureB (gudiv_254), and ureC (gudiv_253). Accessory proteins ureE (gudiv_252), ureF (gudiv_251), ureG (gudiv_250), and ureD (gudiv_249) (C) Diagram of Ureaplasma diversum Multiple-Banded Antigen-like protein (MBA-like—gudiv_653) and locus and similarity of MBA-like with the human ureaplasmal Multiple-Banded Antigen (MBA) (Accession number: AF055358.2).

https://doi.org/10.1371/journal.pone.0161926.g004
Urease.

In addition to the hydrolysis of urea, the enzyme urease can also be a virulence factor of ureaplasmas. The toxicity of urease is mediated by the amount of ammonium ions or free ammonia generated from urea [34]. The urease activity of human ureaplasmas produces ammonia, which can damage host tissues due to changes in the pH [35]. To date, there is no data in the literature on the pathogenic effects of the expression of the urease gene by U. diversum. Nevertheless, the analysis of the organization of the U. diversum urease gene cluster revealed the same organization of the human ureaplasma urease gene clusters [36]. There are three CDSs encoding structural subunits, ureA (gudiv_255), ureB (gudiv_254), and ureC (gudiv_253). Furthermore, downstream of ureC, four CDSs coding for accessory proteins (ureE—gudiv_252, ureF—gudiv_251, ureG—gudiv_250, and ureD—gudiv_249) were found (Fig 4B). A high identity of these CDSs when compared to CDSs of human ureaplasmas was also observed (above 90%).
Phospholipase.

An array of potent hydrolytic enzymes has been identified in mycoplasmas, including phospholipases, proteases and nucleases [37]. Phospholipase C, A1, and A2 (PLC, PLA1, PLA2) activities have been reported in human ureaplasmas, however, no genes showed significant similarity to known sequences of PLC, PLA1, or PLA2 [38]. Orthologs to these CDS, however, were not found in the U. diversum genome. This is somewhat surprising, considering a recent report showing that U. diversum reference strains, including the ATCC 49782 sequenced herein, and clinical isolates have high phospholipase activity [8]. It has been speculated that this phospholipase activity could have helped the in vitro invasion of Hep-2 cells by U. diversum [8]. Furthermore, it has been shown that a similar phospholipase activity of U. diversum are able to interfere with prostaglandin E2 and prostaglandin F2a production by bovine endometrial cells and induce premature labor [18]. In U. diversum, genes encoding phospholipase D family protein (PLD) (gudiv_472) and triacylglycerol lipase (Lipase family, such as phospholipases) (gudiv_748) were identified. Therefore, in contrast to human ureaplasmas, the PLD is likely the true virulence factor of U. diversum linked to cell invasion and premature labor in intrauterine infection, suggesting the phospholipase activity detected in our previous study [8] was PLD rather than phospholipase C activity. Experimental studies should be conducted with PLD to confirm this hypothesis.
Hemolysin.

Hemolysins cause lysis of erythrocytes by forming pores of varying diameters in the host cell membrane [39]. There is one CDS (gudiv_91) coding a putative hemolysin protein in the genome of U. diversum. The predicted protein sequence has 63.1% identity with the hemolysin found in human ureaplasmas. In U. parvum serovar 3, this hemolysin has been characterized as an α-hemolysin (hlyA) with both hemolysin and cytotoxic activities [25]. Other mycoplasmas also have hemolysins [40, 41], but no orthologs to these proteins were found in the U. diversum genome. It seems that the mycoplasma or ureaplasma hemolysins belong to a unique class of bacterial toxins; more studies are needed to better understand the role of this enzyme in the pathogenesis of bovine ureaplasmas.
MIB-MIP system.

Recently, a new virulence factor has been described in Mollicutes: the MIB-MIP system [42]. MIB acts as a high-affinity IgG binding protein, whereas MIP specifically cleaves the IgG heavy chain at an unconventional site, located between the VH and CH3 domains, and inactivate this molecule. This MIB–MIP system is encoded by a pair of genes often found in several copies in a wide range of mycoplasmas that infect various hosts [42]. Accordingly, two copies of the MIB-MIP system (gudiv_161/gudiv_162 and gudiv_612/gudiv_613) were found in the U. diversum genome. However, experimental studies should be conducted to better understand the role of this enzyme in the pathogenesis of bovine ureaplasmas.
Surface molecules.

Although surface molecules of mycoplasmas are thought to play a crucial role in interactions with their hosts, very few had their biochemical function defined [43]. CDSs linked to lipoproteins (n = 37), multiple banded antigen like protein (MBA-like) (n = 1), membrane nuclease lipoprotein (n = 1) and variable surface antigen lipoproteins (possible VsA) (n = 1) were found in the genome of U. diversum (Fig 4A). Possibly due to the absence of a cell wall, mycoplasmas posses a larger number of membrane bound lipoproteins when compared to other eubacteria [44]. These molecules can act as virulence factors and/or be targets of humoral immunity [45]. They serve as potent cytokine inducers for monocytes/macrophages and have cytolytic activity [46]. This immunogenicity of lipoproteins is probably caused by their surface exposure and the presence of the amino-terminal lipoylated structure [47].

Among the detected lipoproteins, one CDS was identified as a membrane nuclease lipoprotein (gudiv_93). Membrane nuclease activity in mycoplasmas was first reported in M. pulmonis [48]. Nuclease activity in Mollicutes has been proposed as the mechanism by which these organisms acquire the precursors for nucleic acid production [49], but it can also function as a virulence factor. The characterization of nucleases in Mollicutes reveals that these enzymes differ from each other [50], and have been implicated in host cytotoxicity. The membrane nuclease MGA_0676, for example, is a remarkable pathogenic compound for the colonization and infection persistence of M. gallisepticum [51]. Additionally, M. pneumoniae nuclease (Mpn133) acts as a virulence determinant by binding to and internalizing within human airway cells [50].

Another important lipoprotein that has been identified is a putative variable surface antigen lipoprotein (gudiv_179) with 36% identity to the VsaA protein of M. pulmonis. Antigenic variation of surface proteins is thought to be a survival strategy for many mycoplasmas [52]. M. bovis possess a large family of variable surface lipoproteins designated as Vsps, which are encoded by 13 different genes. The Vsps undergo phase variation in the respiratory tract of infected calves, which limits the microorganism elimination [53]. The experiments with variable surface proteins support the hypothesis that the generation of antigenic variation in mycoplasmas is critical for the bacterial survival for extended periods within the host, but do not establish a link with disease severity [45].

Although with borderline similarity (33%) a multiple banded antigen like protein (MBA-like) was annotated in the genome of U. diversum ATCC 49782 (gudiv_653) (Fig 4C). This protein showed the conserved domain of the Multiple Banded Antigen-MBA of human ureaplasmas. The MBA is a surface exposed lipoprotein that can undergo size and phase variation in vitro and in vivo [54, 55]. The MBA is predicted to be a major human ureaplasma virulence factor and is the predominant antigen recognized by sera during infections in humans [56]. Analyzing the MBA locus composition among different human ureaplasmas [21], we can verify that the MBA-like locus in U. diversum features a similar organization with the MBA locus of U. parvum serovar 14 (UPA14) (Fig 4C). In particular, this area has two inverted hypothetical proteins and a transposase recognition site. Furthermore, unlike other ureaplasmas, in UPA14 and U. diversum loci, the DNA pol III alpha subunit is not located adjacent to the locus. More studies are needed to understand the biology and role of MBA-like in the pathogenicity of U. diversum. Interestingly, the MBA-like (gudiv_653) and the variable surface antigen lipoproteins (gudiv_179) are in the same paralogous gene family in U. diversum. These two proteins, added to fourteen other paralogous proteins (annotated as hypothetical proteins), may play a critical role in modulating the immune response against U. diversum (S1 Table).
Capsule.

In the analysis of the genome of U. diversum, a glycosyltransferase enzyme (gudiv_216) was found [57]. Glycosyltransferase has also been described in the genome of human ureaplasmas. This enzyme is known to connect lipids and sugars that constitute the capsule of certain mycoplasmas [58]. In M. mycoides subsp. mycoides, the presence of glycosyltransferase is believed to be related to the attachment of galactan to the capsule [57]. Thus, we decided to evaluate if a capsular structure is also present in U. diversum. By using electron microscopy and the red ruthenium dye, a polysaccharide capsule extending from 11 to 17 nm outside of U. diversum cells was observed for the first time (Fig 5A). In a previous study with U. urealyticum, using the same dye utilized herein, a capsular structure was also observed, indicating that this may be a common feature of Ureaplasma species [13]. Increased virulence and adhesion to host cells are associated with the presence of a capsule in certain bacterial species [59]. For instance, the M. gallisepticum and M. hyopneumoniae capsules have been suggested as additional structures that assist the cytoadherence to host cells [60] in addition to the “bleb” (a mycoplasma structure containing adhesins and accessory proteins) [61]. In M. mycoides subsp. mycoides strains, the presence of a polysaccharide capsule causes host injury [62]. Other studies have correlated the presence of a capsule and the resistance of mycoplasma to phagocytic cells. In M. dispar, the capsule has been considered the most important structure to interfere the phagocytosis [63]. Therefore, the detection of a capsular structure in U. diversum opens venues for new studies related to the virulence and pathogenicity of this bacterium.
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Fig 5. Capsule of U. diversum.

(A) Electron microscopy of cells U. diversum ATCC 49782 obtained in the cultured isolates from mucovulvovaginal bovine semen and treated with red ruthenium dye, showing polysaccharide materials (electrodense external region indicated with arrowheads). Bar 100 nm. (B) Percentage of monosaccharides in capsular components of U. diversum ATCC 49782.

https://doi.org/10.1371/journal.pone.0161926.g005

The chemical composition of the capsular material of U. diversum ATCC 49782 was subsequently evaluated. Arabinose, xylose, mannose, galactose and glucose were the main components detected in the capsular material of this bacterium (Fig 5B). The biochemical composition of capsular polymers of Mollicutes has been little studied. The capsules of M. mycoides subsp. mycoides SC, M. mycoides subsp. capri and M. dispar are the only ones described in the literature and are mainly composed of 1,6-galactose, polyglucan, and galacturonic acid polymers, respectively [64]. Probably, the capsular biosynthesis of M. mycoides subsp. mycoides is related to a glucose-dependent synthetic pathway due to the presence of a UDP-glucose 4-epimerase (GalE) and a glucose-1-phosphate uridylyltransferase (GalU) that uses glucose-1-phosphate to generate UDP-glucose [57]. In our in silico analysis, we did not observe genes related to the biosynthesis of polysaccharides. However, the presence of two genes of a sugar transporter was observed (Ribose/galactose ABC transporter—gudiv_307 and gudiv_308). We believe it is likely that these sugars are captured by U. diversum and are incorporated using glycosyltransferase to form the capsule.
Host immune response against surface molecules.

In order to better understand the immune response against U. diversum surface molecules, we infect murine macrophages J774 with viable and nonviable microorganisms. In the analysis of cytokine production, there was an increased production of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) in both conditions compared to the negative control (p<0.05), but there was no statistical difference between the profile triggered by the viable and nonviable ureaplasma (Fig 6A). Therefore, it is suggested that surface molecules are the main determinants of immune system activation in U. diversum. Surface molecules are composed of bacterial pathogen-associated molecular patterns (PAMPs) that, when recognized by the innate immune system, trigger the production of pro-inflammatory cytokines from manifold cells, causing inflammation [65–67]. The ability of surface molecules of Mollicutes to induce the secretion of inflammatory cytokines such as IL-1β, TNF-α and IL-6 have been reported in studies with different species, e.g. U. diversum [5], U. urealyticum [68, 69], and M. fermentans [65].
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Fig 6. Host immune response against surface molecules.

(A) IL-1β, IL-6, TNF-α and IL-10 levels in supernatant of macrophage J774 culture after infection with viable and nonviable U. diversum ATCC 49782 strain compared to uninfected cells. Statistical significance (p<0.05) is represented by the asterisk (*) (non-parametric Mann-Whitney analysis—One-tailed test, GraphPad Prism® version 6.01). (B) Gene expression of Toll-like receptors 2 in macrophage J774 culture after infection with U. diversum ATCC 49782 strain compared to the uninfected cells. Statistical significance (p<0.05) is represented by the asterisk (*) (non-parametric Mann-Whitney analysis—One-tailed test, GraphPad Prism® version 6.01). (C) up-regulated (green) and down-regulated genes (red) of the inflammasome pathways in macrophage J774 culture after infection with U. diversum ATCC 49782 strain compared to uninfected cells. Statistical significance (p<0.05) is represented by the asterisk (*) (non-parametric Mann-Whitney analysis—One-tailed test, GraphPad Prism® version 6.01).

https://doi.org/10.1371/journal.pone.0161926.g006

The expression of Toll-like receptors (TLRs) and inflammasome pathway genes were also evaluated using murine macrophages J774 infected with viable U. diversum cells. It can be observed that U. diversum induced a higher gene expression of TLR2 when compared to the uninfected cells (Fig 6B) (p<0.05). The gene expression of other TLRs are presented in S1 Fig. The downregulation of TLR5, TLR8, TLR9 and TLR11 was als observed. These findings are supported by the literature, which indicates that TLR2 is the main receptor for the immune response of Mollicutes [45]. However, a study of U. urealyticum infecting macrophages demonstrated the activation of TLR2 and TLR4 [70]. Moreover, the absence of TLR 2 was related to the not recognition of bacterial lipoproteins of M. fermentans [71, 72].

The results of the inflammasome expression assay demonstrate the up-regulation of genes related to chemokine binding domains, interleukin-6, inhibitors of kinases and B cells, NF-kB, and interferon regulatory prostaglandin endoperoxide (Fig 6C). In contrast, caspase 12, CD40 ligand, interferon gamma, interleukin 12A, and 33 families of ligand to NLR and tumor necrosis factor genes were downregulated. Several studies have demonstrated the importance of the inflammasomes in response to mycoplasma infection [73]; however, the literature does not define mechanisms involved in the inflammasome response against U. diversum. A study of the development of gastric tumors using monocytes exposed to M. hyorhinis confirmed a high production of IL-1β and IL-18-induced NLRP3-dependent mechanisms [74]. It has been shown that monocytes and macrophages infected with live or inactivated by heat Acholeplasma laidlawii have the capacity to promote the release of IL-1β by the presence of cytosolic ASC (apoptosis-associated speck-like protein containing a carboxy-terminal CARD), promoting inflammasome activation [75]. In the present study, the up-regulation of NLRPs was not observed. Therefore, more studies are necessary to elucidate the involvement of inflammasome activation in the immune response induced by U. diversum. This study is the first report of the activation mechanisms involved in the immune response against Ureaplasma diversum. These findings can contribute to the studies on macrophage activation for the regulated secretion of cytokines to prevent tissue damage caused by an exacerbated immune response. The results described herein will also support further research to understand the possible effector mechanisms that lead to effective protection against U. diversum infection.