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Tai san Nguon von Tai san ngan han a Nqt phai tra Tien mat Vay ngdn han Ngdn hang Tien gui ngdn hang Phdi tra cho ngucri ban f Phdi thu cua khdch hang Thue va cac khoan nop nha nude 50 Hang hod Tai san dai han b Nguon von chu s is hfru e Tai san co dinh Nguon von Kinh doanh Hao mon tai san co dinh Lpi nhuan chua phan phdi g Dau tu dai han khac Quy khen thuang Tong Tiki san c Tong nguon von m B a i ta p 6: A mutation in mrpH , which encodes the fimbrial adhesin, does not decrease fitness in independent challenge, although the mutant was unrecoverable following a one-week cochallenge experiment Figure 7. An abundance of factors pertaining to amino acid transport and metabolism are also upregulated during ascending UTI, including D-serine dehydratase dsdA. Other variations on this assay include the addition of short catheter segments to multiwall plates and culture in static, closed tubes ,

For the six sequenced strains, fimbriae were identified using BLAST using the HI fimbrial major structural subunit gene as the query. Reproduced from , with permission. Despite the overall conservation of the P. A few loci in particular are hypervariable and merit further mention Fig.

Comparative genomic hybridization of Providencia stuartii and Morganella morganii with a P. Further analysis of this 94 kb element, called ICE Pm1 , revealed genes encoding two previously-identified virulence factors: Proteus toxic agglutinin Pta and Nrp siderophore — Furthermore, ICE Pm1 has been observed to transmit between clinical isolates in a laboratory setting Examination of sequenced genomes reveals that ICE Pm1 , when present, is not always inserted at the same genetic locus.

Specifically, the genes encoding the T6SS secretion apparatus are highly conserved, but the secreted effectors vary in sequence, type, and number. For example, P. In both cases, the effector operons begin with hcp and vgrG homologs, which are required for T6SS function Furthermore, HI encodes at least two more operons identified as likely T6SS effectors than BB is predicted to encode , Examination of other P.

The rest of the operons are not homologous black. The scale at the top is in nucleotides. Like other gram-negative bacteria, P. In a survey of P.

Specifically, strains with negatively-charged O-polysaccharides displayed higher urease, protease, and swarming activities compared with positive or neutral O-polysaccharides LPS is abundant on the cell surface of gram-negative bacteria, and its properties can affect bacterial interaction with their environment. Thus, some aspects of strain variability may be determined by LPS biochemistry. Flagella link flagella section are targets of the innate immune system and also energetically expensive to produce Correspondingly, although most flagellar genes are conserved in P.

The properties of FlaAB hybrids have been extensively reviewed elsewhere Like other bacteria, P. HI encodes three apparently complete and three degenerate prophages HI and BB , Differential expression of phage genes has been observed in P. Furthermore, phage have been used to transduce swarming ability into non-swarming strains Thus, it is likely that phage account for some of the observed differences in swarming behavior between strains.

Plasmids are not a defining feature for P. The original P. However, several of the targeted genes were involved in plasmid stability, and pHI does not encode any obvious virulence genes Global analysis of gene expression transcriptomics has been conducted for P. The four following studies , , , were conducted using a microarray with mer oligonucleotide probes designed to hybridize with each of the 3, predicted open reading frames from an early draft version of the P.

Perhaps the most dramatic change P. This transformation was the target of the first P. The data were consistent with earlier reports of increased expression of certain virulence genes during swarming , , and swarmer cells being less metabolically active than consolidates Curiously, although P. Based on the accumulated transcription and metabolic data, the consolidation phase has been proposed to be a time of preparation for the next round of swarming, by increasing nutrient uptake systems, central metabolism, and respiration.

Iron is sequestered by the host to combat bacterial infection, and pathogens activate iron acquisition systems during UTI link metals section. However, microarray analysis conducted on iron-restricted P. A critical point for studying how P. To answer this question, urine was collected from experimentally infected mice at 1, 3, and 7 days post-inoculation dpi for RNA extraction and microarray hybridization Iron acquisition, urease, and peptide transporters were induced during UTI, consistent with an iron-restricted, urea- and peptide-rich urine environment.

However, some known virulence factors were either poorly expressed or downregulated during UTI, including genes encoding PMF fimbriae, Zap metalloprotease, hemolysin, and Proteus toxic agglutinin Pta. This suggests that voided urine provides an important, but incomplete, snapshot of bacterial gene expression during UTI.

A Heat map of expression data for specific virulence-associated genes, depicting the ratio of expression in LB broth versus in vivo.

The legend at the left indicates the color associated with log 2 fold change: B Adherence and motility genes are inversely regulated during UTI. Each line represents fold-change of a specific flagellar left panel or fimbrial right panel gene in vivo relative to mid-logarithmic phase culture in vitro. Genes in the mrp operon are highly induced early during infection, but expression falls by 7 days post infection.

Flagellar genes are initially repressed, but expression increases late in infection. Interestingly, central metabolic pathways that were required during infection differed in several ways compared with a previous study of uropathogenic E. Nitrogen assimilation gene expression suggested that P. In contrast with E. Gene expression at 1, 3, and 7 days post-inoculation was also compared. Flagellar genes were initially repressed, but this repression was relieved by day 7.

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In contrast, mrp genes were highly-transcribed throughout the infection but their degree of induction was lower at day 7; this topic is further detailed in the fimbrial section link fimbriae section. The transcriptional regulator MrpJ is encoded by the mrp fimbrial operon link fimbriae section.

Originally proposed as a switch between adherent and motile states , MrpJ was found to contribute to other phenotypes as well MrpJ and MrpJ paralogs are discussed later in this chapter link.

The next three studies , , compared gene expression for targeted mutants with their isogenic parent strain using RNA-seq. In addition to the aforementioned microarray study, broth-cultured and swarmer cells have also been compared by RNA-seq using the BB lab strain Even though the methods for collecting and analyzing swarmer cells were different in these two studies , , similar results were obtained for the broth-swarmer comparison regarding the genes encoding flagella, lipoproteins, iron transport, and hemolysin, among others.

FliL is a flagellar protein that, when mutated, leads to elongated swarmer-like cells under non-swarming conditions pseudoswarming Comparison of a fliL mutant cultured in broth to induce pseudoswarmer cells with the wild-type broth RNA-seq dataset revealed an increase in propanediol utilization in the fliL mutant.

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This suggests a possible new role for these genes in swarmer cell differentiation The Rcs phosphorelay is a repressor of swarming and an activator of biofilm formation , , — RNA-seq was used to compare a broth-cultured rcsB mutant with its isogenic parent strain PM , In this condition, the rcsB mutant undergoes inappropriate elongation that is similar to the fliL phenotype. These studies showed that RcsB controls pathways important for swarming, including cell division minCDE and motility flagella and flagellar regulators.

The first targeted mutation to be made in P. This mutant was constructed using a single crossover, Campbell-type insertion via an R6K ori-pir suicide vector introduced by conjugation , an approach that is still used by multiple groups studying P.

This initial paper noted several difficulties with methods for mutant construction in P. Later on, sacB counterselection was employed to facilitate identification of double crossover events The ability to directly construct and test mutants in the mouse UTI model was a boon for dissecting P.

Even so, mutant construction remained labor-intensive and prone to failure, particularly in some clinical isolates. Integration of pBDJ into the chromosome was selected on medium containing tetracycline and ampicillin. Transconjugants Ap r Tet r were urease negative. The 1. Adapted from 40 , with permission. Thus, another method was developed for constructing mutations, using a group II intron mutated to specifically target a gene of interest , Advantages over the prior allelic exchange method are the relatively short time required, higher rate of success, and lack of passaging or counterselection that would normally be required to resolve double crossover events.

The drawbacks are that the choice of insertion sites is limited, mutations have polar effects on downstream genes, and the method is insertion-based that is, it is not simple to delete sequences. The latter problem can be managed by use of cre -lox recombination to remove the antibiotic resistance cassette carried within the intron, resulting in an unmarked mutant Additional introns can be inserted, with the potential to remove intervening sequences via an additional round of cre -lox recombination.

Genes that have been specifically mutated and tested in a mouse model of UTI, either independently or by direct cochallenge against a parental strain, are shown in Table 4.

It is important to note that the studies presented in Table 4 include a range of P. Transposon mutagenesis in P. This approach has since been used to generate libraries for isolation of mutants defective for a given phenotype, such as swarming motility , , — , sensitivity to antimicrobial peptides 80 , , and biofilm formation In , this approach was revolutionized through the development of signature-tagged mutagenesis STM , in which each transposon mutant has a specific DNA sequence within the transposon that acts as a barcode.

STM led to the first genome-wide pathogenesis studies by permitting identification of unique mutants within a mixed population and allowing for assessment of mutants lost during the selective process of the infection, and therefore containing transposon insertions in genes that contribute to colonization and pathogenicity.

This approach was successfully utilized three times with P. P, primary screen; U, urine; B, bladder; K, kidney; O, attenuated overall. In the first P. Only two mutants were reproducibly underrepresented in the output samples from all five mice: The fitness defect of each mutant was assessed by direct cochallenge with wild-type P.

The second P. A secondary screen was conducted with these mutants divided into 19 pools, and only of the mutants were reproducibly attenuated. Nucleotide sequences were obtained from 30 of the mutants, 27 of which map to open reading frames in the current annotation of P. This approach identified several categories of mutants representing both known and novel fitness factors for UTI, including motility, iron acquisition, plasmid-encoded factors, transcriptional regulation, phosphate transport, urease activity, capsule synthesis, and metabolic pathways, the majority of which would have been difficult to identify as fitness factors by other methods.

The third P. Nucleotide sequences were obtained from 29 of the mutants, 28 of which map to open reading frames in the current annotation of P. This approach again identified fitness factors pertaining to cellular processes, transport, transcriptional regulation, motility, cell surface structures, and metabolism. In , the basic technique of STM was further improved upon and integrated with next generation sequencing techniques to allow for massively parallel sequencing and quantitation of the relative abundance of all mutants present in a given setting.

Several variations of the method were developed concurrently, including insertion sequencing INSeq , transposon-directed insertion site sequencing TraDIS , transposon insertion-site sequencing Tn-Seq , , and high-throughput insertion tracking by deep sequencing HITS As Tn-Seq has been applied to the study of P.

The first step of Tn-Seq is to generate a genome-saturating transposon mutant library, generally using a Mariner transposon. Due to the saturating nature of the library, this method can also be used to identify genes that are essential for growth in vitro , as insertion mutants within these genes will be lacking from the starting library. Infection studies are then conducted with the transposon library, and all bacteria in the target site of infection for instance, the bladder are collected for extraction of genomic DNA.

The frequency of each insertion mutant is then determined, en masse , from the input sample the saturated library and the output sample the bladder , and the fitness contribution of each gene in the genome can be assessed by the change in frequency of insertion mutants for that gene from the output sample compared to the input sample. This approach was successfully used to generate the first genome-saturating library of transposon insertion mutants in P.

The transposon mutant library was then used in a murine model of CAUTI to identify fitness factors for colonization of the catheterized bladder and ascension to the kidneys 4 days post-inoculation Fig. Tn-Seq therefore provided confirmation of a role for known P. For each of five transposon mutant library pools, mice were infected as follows: Thus, for each input pool, the single-species infections and coinfections were conducted in parallel to utilize the same input inoculum.

Input and output samples were enriched for transposon-containing sequences and subjected to next generation Illumina sequencing of the transposon-chromosome junctions.

The resulting reads were mapped to the P. The gene in yellow represents a candidate P. Importantly, this approach was also applied during polymicrobial infection with P. Of the genes identified as P. The fitness factors that were important for both infection types include fimbrial genes, components of the flagellar cascade, urease, and factors involved in inorganic ion transport and metabolism. Interestingly, an additional 1, candidate fitness factors were identified that appear to specifically contribute to P.

It was further determined that the requirement for BCAA biosynthesis during coinfection was due to high-affinity import of leucine by P. Urease is a nickel-metalloenzyme, the synthesis of which is induced by the presence of its substrate, urea , To become catalytically active, the apoenzyme must acquire divalent nickel ions through a process involving four accessory proteins, UreD, UreE, UreF, and UreG. The three-dimensional structure of urease, inferred from the closely related urease of K.

Although UreD and UreF interact in the absence of structural proteins, UreD is still capable of associating with the apourease without coaccessory proteins such as UreF. These interactions could stabilize the accessory protein interactions with the apourease and hypothetically coordinate nickel uptake among the three active sites of urease.

C The 6,bp P. In early studies using bacterial cell lysates of four P. The affinity for substrate as estimated by K m is relatively weak, ranging from 22—60 mM urea among the four isolates. The urease operon was originally characterized from a DNA fragment of 7. Six open reading frames were found within a 4, bp region, which were predicted to encode polypeptides of Reexamination of recombinant clones for urease activity revealed that a seventh gene, ureG , was also required for production of catalytically-active urease enzyme Sequences that preceded these genes later determined to confer urea-inducibility upon the operon , resulting in identification of UreR, a UreR binds to these sites in a urea-dependent manner to activate expression of the urease operon.

In the absence of urea, there is no measurable binding or induction of urease expression. Using a ureR-lacZ reporter plasmid, it was determined that H-NS represses transcription of ureR , and therefore represses urease expression , H-NS and UreR compete for binding the ureR-ureD intergenic region, and culture conditions dictate whether the operon will be repressed or activated. Urease is a critical virulence determinant for P. Indeed, the first targeted mutation of this species involved the mutation of ureC , the major structural subunit of the apoenzyme The ID 50 of the ureC mutant is approximately 3 logs higher than the parental strain, clearly indicating the contribution of urease to disease severity.

In an independent challenge with 10 9 CFU of the wild-type strain or the ureC mutant, the mutant was attenuated by approximately 2 logs in the bladder and kidneys hours post-inoculation and up to 6 logs by 7 days post-inoculation 39 , Infection with the ureC mutant also resulted in significantly less pathology in the bladder and kidneys of infected mice. The contribution of urease to pathogenicity has also been assessed in the uncomplicated murine model of UTI compared to a model of catheter-associated UTI CAUTI , in which a catheter segment is placed in the bladder during inoculation These studies determined that urease significantly contributes to cystitis in both models, but does not play as dramatic a role in pyelonephritis in the CAUTI model.

Urine colonization in catheterized individuals is frequently polymicrobial, and P. It is therefore noteworthy that co-culture with other uropathogens can enhance production of active urease enzyme by P. In addition to P. This has important implications for the pathogenesis of polymicrobial CAUTI, as enhanced urease activity resulting from co-culture of P.

Due to the important role of urease during P. Such inhibitors include hydroxyamic acids, which bind the nickel atoms in the urease active site, and acetohydroxamic acid AHA , a structural analog of urea. AHA is the only analog to have been tested in clinical trials and be approved by the Food and Drug Administration.

However, while it showed efficacy for preventing urolithiasis — , it has potentially severe side effects that limit its clinical use Thus, further research is necessary to identify safe and effective urease inhibitors. It is also worth noting that while the ure operon is urea-inducible, structural analogs of urea such as AHA do not induce urease expression These pathways are predicted from the genome sequence , as analyzed by KEGG pathways and from the individual studies described below.

While examples of secreted proteins can be found for the systems noted, only a few examples of secreted proteins that have been tested for their contribution to virulence have been documented.

A correlation between the ability to swarm and protease secretion was noted. These proteases were all EDTA-sensitive metalloproteinases However, the specific secretion systems involved in secretion of these proteases were not determined in this study. Nucleotide sequencing of the genome of P. A cluster of 24 genes clearly have the potential to encode an intact T3SS with genes for an intact needle complex and at least two effector proteins and their chaperones.

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The genetic organization of the T3SS is similar to that of Shigella flexneri , there was no evidence of mutation of the genes resulting in inactivation of the system, and RT-PCR analysis demonstrated that these genes were expressed in P. However, mutation of two genes including a putative ATPase and negative regulator of T3S had no effect on the secreted protein profile when compared to the wild-type strain Furthermore, in the murine model of ascending UTI, there was no difference in CFUs between the mutant and wild-type strain in urine, bladder, or kidneys during either independent infection or cochallenge.

Thus, no pathogenicity phenotype for the T3SS was identified for P. There are three subclasses of type V secretion systems Va, Vb, and Vc , and the nucleotide sequence of P. Classical autotransporters are a family of virulence proteins in Gram-negative pathogens that contain three domains: One of the three predicted classical autotransporters encoded by P.

Proteus toxic agglutinin pta , encodes a serine protease that was previously identified as an immunogenic outer membrane protein link Toxins section. The kDa protein has a amino acid long signal peptide, a kDa long N-terminal passenger domain, and a kDa C-terminal translocator The autotransported protease, either cell-associated or secreted, has cytotoxic effects on cultured bladder and kidney epithelial cells.

Catalytic residues Ser, His, and Asp, when mutated, abolish protease activity. The protein also has autoaggregation properties not associated with the proteolytic activity. Virulence of a protease mutant, as tested in the murine model of ascending UTI, was significantly reduced.

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This requires sequential unfolding of the HpmA protein , for which a partial crystal structure has been solved Two polypeptides, HpmB and HpmA, synthesized in that transcriptional order, are responsible for hemolysin activity of P.

HpmB is necessary for secretion of HpmA. Recombinant AipA and TaaP bind extracellular matrix proteins, produce polypeptides of kDa and kDa, respectively, and form high molecular weight homotrimers.

A amino acid invasin-like motif of AipA is necessary for function. The discovery of T6SSs in P. The Dienes phenomenon, first described in by Louis Dienes, refers to the fascinating ability of two P.

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Formation of the Dienes line requires direct cell-cell contact by living bacteria, and involves killing of at least one strain at the boundary Fig. Loss of the T6SS in both isolates allows non-identical swarms to merge and the lack of T6SS-dependent killing appears as recognition white arrow. A visible boundary formed between swarms of the wild type and the deletion mutant.

Swarms of the wild type merged regardless of the presence of pKG The bar shown is 1 cm. Figure adapted, with permission, from A and B. Strain 3G produces round cells, whereas strain 2R produces no round cells.

The dark areas are agar with no growth. Work conducted in P. Further investigations in P. Using a transposon screen for P. This operon was found to encode a single immunity protein, PefE, which was responsible for providing immunity from killing by the Pef effectors.

Differential use of these systems or possibly even recombination between them may therefore contribute to the wide range of strain-killing activities and Dienes types exhibited by P.

A A circular representation of the P. Genes with homology to hcp grey , vgrG white , and predicted T6SS effectors blue are shown.

A direct role for any individual P. For instance, none of the T6S operons encoded by P. However, all of the known P. It remains to be determined if this system is important for mediating microbe-microbe interactions or microbe-host interactions in this infection model.

While the complete genome sequence of P. These are hemolysin, Proteus toxic agglutinin, and the ZapA metalloprotease. Secreted pore-forming toxins are a common feature of pathogenic bacteria.

In particular, hemolysins are secreted pore-forming toxins that insert into eukaryotic cell membranes, causing efflux of sodium ions and cell damage The calcium-independent hemolysin is encoded by two genes comprising a two-partner secretion system: HpmA is the predominant hemolysin in Proteus species and appears to be the only hemolysin encoded by P.

Similarly, hpmA is present in all 7 complete P. This fragment lacks the C-terminal pore-forming domain, but can still activate full-length HpmA in the absence of HpmB when the proteins are mixed with erythrocytes. The formation of HpmA dimers suggests that full-length HpmA may also function as a dimer. HpmA mediates lysis of a broad range of cell types from numerous host species and appears to be the primary P.

Deletion of hpmA dramatically decreases cytotoxicity, and allows for internalization of P. As HRPTECs form a protective barrier for the kidney parenchyma, it was hypothesized the hemolysin may be a critical virulence factor that mediates spread of P. However, deletion of hpmA does not appear to impact tissue colonization or damage during independent challenge in the murine model of ascending UTI 45 , 48 , 55 , indicating that either hemolysin has less of an impact during experimental infection than the in vitro cell culture studies suggest, or the activity of other virulence factors mask its contribution in vivo.

In agreement with this finding, hpmBA was not identified as a fitness factor for UTI or CAUTI in any of the genome-wide transposon mutagenesis studies , , , , likely due to a combination of complementation in trans by the other hemolysin-producing transposon mutants present during infection and possibly production of other cytolysins with similar functions.

The genome of P. Pta was initially identified as an outer membrane surface-expressed protein recognized by the murine immune system , and determined to be encoded within the integrative and conjugative element ICE Pm1 in P. Expression of this protein in E. Both properties of Pta require translocation to the outer membrane and cytotoxicity requires serine protease activity from the passenger domain, but protease activity is not required for autoaggregation.

Pta is also classified as a subtilisin-like alkaline protease owing to the observation that expression of pta is induced by alkaline pH and protease activity is maximal at pH 8. Pta contributes to colonization of the bladder and kidneys in a murine cochallenge model of ascending UTI, as well as dissemination to the spleen Interestingly, HpmA and Pta have an additive effect on cytotoxicity both in vitro and during experimental UTI, particularly with respect to cystitis and possibly interstitial nephritis However, Pta appears to be the more potent toxin during experimental infection, as disruption of pta has a much greater impact on infectivity than loss of hpmA.

Similar to hpmA, pta is present in all seven of the complete P. In all cases, cleavage was sensitive to the metal chelator EDTA, indicating that the enzyme is a metalloprotease. This protease was identified as a 55 kDa metalloprotease of the serralysin family, encoded by zapA , The contribution of ZapA to pathogenicity has been explored both in vitro and in vivo. Intact IgG and IgA1 can interact with Fc receptors on neutrophils to stimulate a respiratory burst, and this process is significantly reduced when IgG has been cleaved into Fab and Fc fragments in vitro by the P.

Thus, ZapA may contribute to evading the innate immune response during infection. A ZapA-deficient mutant, specifically constructed by insertional inactivation of zapA in the chromosome of P. After 7 days, quantitative cultures indicated that the zapA mutant was dramatically attenuated by 3 logs in the urine and 5 logs in the bladder or kidney , indicating that the protease contributed strongly to virulence of P.

The same wild-type and mutant pair were tested in a rat model of prostatitis Unlike the wild-type, inoculation of the zapA mutant resulted in reduced levels of acute prostatitis as determined by lower levels of tissue damage, bacterial colonization and inflammation Fig. Pathogenicity islands PAIs are a specific group of genomic islands that contribute to genomic variability and virulence of bacterial pathogens.

PAIs carry virulence determinants and are typically present in pathogenic strains but absent in nonpathogenic strains of the same species. They also frequently contain mobile genetic elements, are flanked by direct repeat sequences, have mosaic-like structure, insert at the site of tRNA genes, and have likely been acquired by horizontal gene transfer , A subset of PAIs has been identified that can excise from the bacterial chromosome, following a recombination event at the site of direct repeats flanking the island, and actively transfer via a type IV secretion system to another bacterium.

In the genome of P. Modules in yellow represent core modules; variable regions are depicted in gray. Direct repeats DRs are represented as triangles. Boundaries of ICE Pm 1 are denoted by vertical black lines. DRs show the modularity of the island and suggest the evolutionary history of acquisition of regions of ICE Pm 1. ICE Pm 1 contains 91 ORFs, with the core genes constituting putative integration, replication and conjugative modules, including an integrase, genes for a putative tyrosine-like recombinase, putative helicase that could act as a relaxase, six transposases, and five plasmid-transfer related proteins A gene region encodes eight putative exported proteins and nine putative membrane proteins comprising the T4SS.

These cargo genes include the nrp operon PMIPMI , which encodes genes for the synthesis, transport, and uptake of an iron siderophore link metals section , and Proteus toxic agglutinin Pta , which contributes to aggregation of P. ICE Pm 1 was also found at a reduced frequency in colonizing isolates of P. When the ICE-encoded integrase is activated, the ICE can excise from the chromosome, form a circular intermediate, and subsequently transfer to a recipient cell via a mating pore formed by the ICE-encoded T4SS , Transfers occurs at a frequency of 1.

Insertional inactivation of a putative integrase gene PMI , a specific recombinase of the tyrosine-like family, on ICE Pm 1 decreases transfer frequencies to below the limit of detection. Mutation of the relaxase of ICE Pm 1 also eliminates transfer Fimbriae pili are hair-like protein structures that extend from the bacterial surface and usually mediate adherence to surfaces.

Gram-negative bacteria produce a subset, called chaperone-usher fimbriae for their method of secretion and assembly , Although the function of most of these is not yet defined, transcription has been detected from all 17 operons, and most of these fimbriae are encoded by P.

Of seven clades defined for classical chaperone-usher fimbriae, three are represented in P. Table adapted from For example, at least two fimbrial morphologies have been seen on a single bacterium by transmission electron microscopy , This ability sets P. Likewise, P. Because fimbriae are often found to contribute to virulence, there are multiple studies examining the roles of P. In general, mutations in individual fimbrial genes result in bacteria that are still able to colonize the urinary tract, but are less fit in direct competition cochallenge experiments , , This suggests that the 17 fimbriae have overlapping or redundant functions.

Furthermore, as surface-localized, abundant, immunogenic proteins, P. These are the most-extensively studied of the fimbriae encoded by P. This was in contrast to mannose-sensitive agglutination, e. The mrp gene cluster consists of two transcripts. The last gene, mrpJ , encodes a transcriptional regulator and is discussed later. The second transcript comprises a single gene, mrpI , which is divergently transcribed from the rest of the mrp genes The transcriptional start site lies within the invertible element , and thus the orientation of this element dictates whether or not mrp fimbrial genes are transcribed.

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The orientation of the element may be easily detected using PCR-based assays Fig. A Urine, bladder and kidney samples were collected at 7 dpi from mice infected with wild-type P. Note that the top left bacterium is gold labeled, while the top right bacterium is unlabeled.

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Scale bar, nm. Bladders from mice challenged with P. Figure adapted, with permission, from A and B and C. The nine genes in the mrp operon are the most highly induced genes by bacteria in urine collected from experimentally-infected mice compared with in vitro broth culture Correspondingly, even when mice are infected with P.

That is, an mrp locked on mutant outcompetes locked off in broth, and, at the same time, mrpA expression increases during oxygen limitation Transcription of the recombinase mrpI may also be regulated by oxygen levels Assembly requires disulfide bond formation, and mutation of the gene encoding thiol: Interestingly, phase variation contributes to biofilm development.

In contrast, the locked on biofilm is short, very dense, and lacks channels. It is possible that these two studies were in fact not examining the same fimbria A and C The wild-type—infected bladder shows a regional view of clusters, whereas the mutant-infected bladder shows a close-up of the urothelial surface.

Scale bars, in micrometers, are as marked. B and D Alizarin Red staining of bladder sections. Only wild-type P. L, bladder lumen; an asterisk indicates an extracellular cluster.

Arrows indicate regions with increased DAPI signal. Figure adapted, with permission, from 44 A—D and E and F. The magnitude of deficiency of an mrp mutant in mouse model independent challenges varies from study to study, from the initial mrpA experiment, where a 6—fold decrease was observed in urine, bladder, and kidneys, to a four log defect in the bladder for an mrpG mutant , , A mutation in mrpH , which encodes the fimbrial adhesin, does not decrease fitness in independent challenge, although the mutant was unrecoverable following a one-week cochallenge experiment Subsequent cochallenge infections with an mrpA or an mrpA-D mutant likewise resulted in the mutant being significantly out-competed in both the bladder and kidneys , Two studies have reported equal numbers of recovered bacteria from the bladders of mice infected with wild-type compared with an mrpA mutant 44 or mrp locked-off Notably, both of these studies used a relatively early endpoint 24 hpi 44 , compared with other mouse independent challenges lasting 7 days , , , Thus, the location of P.

Second, the percentage of bacteria with the mrp invertible element in the on orientation directly correlates with the amount of bladder colonization Fig. Third, when a locked-on mrpI mutant is competed against wild-type bacteria in a 7 d mouse cochallenge experiment, locked-on bacteria outcompete wild-type in the bladder but not the kidneys Fourth, P. Fifth, wild-type P. A fimbrial preparation from P.

The importance of this protein, UCA, in uroepithelial cell adhesion was subsequently confirmed when a mutation of the gene encoding the major structural subunit of UCA ucaA was tested with desquamated cells Expressing cloned uca genes in E.

However, no fimbriae were observed on the recombinant E.

Although UCA fimbriae are encoded by most P. Antiserum raised against purified fimbriae further indicated that UCA is widely present but variable in size It is possible that variations in UCA may explain distinct phenotypes reported by different groups, who are typically using locally-obtained isolates in their experiments.

Notably, the uca operon is part of a mobile element and flanked by phage genes; in the first two fully-sequenced P. The possible receptors have not been identified in healthy bladder tissue, although galectin-3 is a marker for bladder cancer Thus, it is not apparent whether these molecules are true in vivo targets for UCA.

Given that bladder cell adherence is a defining feature of UCA, it is perhaps paradoxical that a ucaA mutant is significantly attenuated in the kidneys but not the bladder during either independent or cochallenge of the murine urinary tract Transcription of ucaA has been detected in urine from infected mice, although the level was 2. PMF fimbriae were initially identified when a P. The defect was more profound in cochallenge, where the mutant was significantly attenuated in both bladder and kidneys In a hematogenous model, where P.

In summary, evidence exists for PMF to contribute to infection in the bladder, kidneys, or both, and the receptor s have not yet been identified. This suggests that PMF fimbriae may be transiently produced during infection, or that PMF-producing bacteria are not readily voided in urine. This is consistent with overlapping roles for the P.

A and B Independent challenge. The double mutant is significantly less fit compared with either single mutant. C and D Mice were challenged with a 1: Each dot represents the log 10 CFU recovered from each organ. The median horizontal bar is indicated for each group. The range of detection in this assay is 10 2 to 10 9 CFU per organ. Figure adapted, with permission from ATF fimbriae were first identified in a preparation of sheared surface proteins derived from an mrpA mutant The atf operon was originally reported to consist of three genes, atfABC as isolated from a cosmid clone, but subsequent genome sequencing revealed three additional genes atfDEF including an mrpJ paralog In a similar vein, an atfA mutant is recovered at the same rate as wild-type from either independent or cochallenge mouse studies However, when an mrpA mutant was examined by immunofluorescence microscopy in infected mice, AtfA was readily detected Fig.

ATF may contribute to biofilm formation under some conditions Green, GFP-expressing P. This fimbria was first discovered in a sheared protein preparation from a canine UTI isolate of P. The pmp operon was also reported to be directly regulated by cyclic AMP receptor protein Crp Fimbria 14, so named because it is encoded by the fourteenth of the 17 chaperone-usher fimbrial operons in HI, seems like it should be non-functional because the fim14 operon completely lacks a chaperone and has a frameshift mutation in usher gene fim14C However, putative minor fimbrial subunit fim14B was detected in an STM screen for genes that contribute to UTI , and putative major structural subunit Fim14A was identified in a preparation of sheared surface proteins Although chaperone-usher secretion apparatuses are usually specific for a single fimbrial type, it is likely that Fimbria 14 is assembled using one or more of the other 16 chaperone-usher systems encoded by P.

The remaining fimbriae encoded by P. This ability is variable for P. Some of the 17 fimbriae are more variable in sequence than others. For example, one operon, fim3 , is present in less than half the isolates in a diverse collection Likewise, UCA is widespread in P. Transcriptomic and high throughput sequencing methods are beginning to shed light on the complex collection of the 17 fimbriae. Transcripts from three additional fimbriae, fim5, fim8 , and fim15 , were detected by microarray analysis of urine from infected mice, although both fim5 and fim8 were decreased compared with in vitro culture In addition, Fim8A, E, and F protein were detected by mass spectrometry from sheared surface proteins produced during in vitro culture Southern blot analysis suggested P.

MrpJ is a transcriptional regulator encoded at the end of the mrp fimbrial operon Like other mrp genes, it is not expressed well in vitro , but is among the most highly-induced genes in urine collected from infected mice That is, expression of mrpJ on a plasmid represses FlaA flagellin, in turn inhibiting both swimming and swarming motility Fig.

The three strains assayed here are P. A The three strains were assayed for swarming on 1. B Three overnight Luria broth cultures of each of the three strains were adjusted to the same optical density, and equal volumes processed for SDS—PAGE and subsequent western blot analyses with antiserum against MrpJ or P.

It is a member of the xenobiotic response element XRE superfamily of transcriptional regulators. However, deletion of the C-terminal domain has almost no effect on motility repression, suggesting that this region is disposable for reciprocal regulation Consistent with its role as a repressor of motility, MrpJ directly binds the promoter of the master flagellar regulatory genes flhDC , In addition to influencing motility, MrpJ also appears to be involved in autoregulation of the mrp operon.

Expression of mrpJ on a plasmid results in elevated mrpA expression , and an mrpJ mutant produces less MrpA protein The mrpJ mutant also has the mrp invertible element overwhelmingly in the off orientation, even under mrp inducing conditions Deletion of mrpJ has little effect in vitro , which may be due to either low expression of mrp genes in most laboratory conditions or masking of MrpJ function by redundancy see MrpJ paralogs below However, an mrpJ mutant is significantly outcompeted during mouse independent and cochallenge experiments , , indication a strong contribution to fitness and pathogenesis within the urinary tract.

Because mrp genes are transcribed at a low level in vitro , an in vivo mimic has been used to study MrpJ targets, where mrpJ is expressed in trans at levels comparable to those detected in mice during experimental UTI In a comparison of the in vivo mimic with a vector control, genes were differentially regulated. Because many MrpJ-regulated genes have known or predicted roles in disease, MrpJ has been proposed as a master regulator of virulence.

Chromatin immunoprecipitation followed by PCR ChIP-PCR indicated that MrpJ binds within the invertible element of the mrp operon, in agreement with detection of an MrpJ-responsive element — bp upstream of the transcriptional start site of the mrp promoter However, a distant site of action for MrpJ, relative to other transcriptional regulators, is consistent with the flhDC promoter binding data mentioned above , Genomic sequencing of P. All but four are part of fimbrial operons, and they are always located at the beginning or the end of the operon Table 6.

Expression of all but two in trans causes repression of motility Apparently, the paralogs do not all repress motility by the same mechanism, because overexpression of individual paralogs leads to flagellin levels ranging from wild-type to near-complete repression Fig.

Different paralogs also induce distinctive swarming phenotypes and uniquely aberrant differentiation into swarmer cells Fig. This suggests that although most MrpJ paralogs repress motility, they likely have other, non-overlapping functions. A Western blot of flagellin expression in P.

Lysates were also blotted with anti-UreD antibody as a loading control lower panel. Molecular weight markers are indicated on the left side in kDa. B Representative swarming phenotypes of strain HI expressing mrpJ paralogs. C , Gram stains of bacteria taken from the edge of swarm fronts. Notably, MrpJ paralogs are not restricted to P. Because fimbriae allow bacteria to adhere or form biofilms in specific environments, and thus respond to niche-specific signals, MrpJ paralogs have been proposed to orchestrate genetic programs that are beneficial to those environments , Like MrpJ itself, the MrpJ paralogs regulate other fimbrial operons in addition to repressing motility Strikingly, AtfA and Fim8A strongly induced their respective operons while having modest or no effects on other fimbrial genes mrpA , ucaA , or pmfA ; in contrast, UcaJ has no effect on the uca operon Examination of the atf promoter using deletion analysis indicated that AtfJ interacts with a region to bp upstream of the transcriptional start site Thus, other MrpJ paralogs may share a common feature of binding DNA far away from transcriptional machinery binding sites.

Deletion of the AtfJ C-terminal tail had little effect on motility repression, but was required for atf autoregulation Protein modeling of the AtfJ C-terminus suggests this region is involved in protein-protein interactions, and it has been proposed that MrpJ paralogs exert their unique properties via their divergent C-termini , These potential, unidentified, binding partners may explain how MrpJ paralogs exert transcriptional control from distant DNA binding sites.

Biofilms, which are adherent microbial communities, are a notorious problem on catheter surfaces, including urinary catheters.

Less well-understood is the potential establishment of biofilms within the urinary tract, and to what extent these biofilms contribute to disease. Because catheterization is a major risk factor for P. In the presence of urine, struvite and apatite minerals are deposited among the developing P.

In laboratory models, crystalline biofilms form within 6 h after inoculation with P. This process occurs as a result of urease activity link urease section , which causes locally-increased pH and subsequent mineral precipitation.

This trait is a major reason why P. In both these cases extensive crystalline material can be seen occluding the catheter lumen. Random mutagenesis to find biofilm mutants indicates that, as in other bacterial species, the biofilm lifestyle for P. When a subset of these mutants were further examined, most were also deficient in swimming and swarming motility.

Notably, swimming and swarming may be involved in both establishment of biofilms, as a means of rapidly colonizing a surface, and dispersal into new environments. However, motility may also interfere with establishing adherent communities , and it is likely that balance is required for fully-developed biofilm formation and swarming behavior. Notably, P. Infection stones and crystalline biofilms are both dense, complex bacterial communities, and at least two P.

During experimental UTI in mice, P. In both cases, the bacteria may be protected from immune responses 44 , Other processes also contribute to P. Surface hydrophobicity, mediated by lipopolysaccharide LPS 53 , , and capsular polysaccharides — , influence both initial surface colonization and biofilm maturation.

Multiple groups are developing new approaches to prevent biofilm establishment, including alternative catheter materials, antimicrobial coatings or instillation of anti-biofilm chemicals, mechanical or electrical biofilm dispersal, bacteriophage, and control of urinary pH , — Thus far, no catheter type has been immune to P. In some cases, P. Ultimately, a combination of these strategies may be necessary to successfully combat P.

An important caveat for biofilm studies in general is that biofilms can be generated using many techniques, and the chosen method has a strong influence on the outcome of the study A common, simple method for surveying biofilm formation involves culturing bacteria in multi-well plates, and visualizing biofilm by staining with crystal violet.

The major advantage is that this assay allows for simultaneous screening of many samples; it is also easily quantifiable by dissolving the crystal violet and measuring the dye with a spectrophotometer. For this reason, the assay has been used to screen P. Other variations on this assay include the addition of short catheter segments to multiwall plates and culture in static, closed tubes , It is, however, important to note that these methods have their limitations.

While urinary catheter biofilms are subject to the flow of urine, bacteria in multi-well plates are in a closed system where nutrients are depleted. Furthermore, if urine artificial or real is used as the medium, P. This lethal effect in static systems can be seen in a comparison of P. Therefore, while the crystal violet method is useful, results may be less likely to lead to translational applications. Continuous flow systems, where fresh medium is supplied at a constant rate, allow study of P.

The flow can be adjusted to match physiological urine rates in humans, and a flow cell can be inserted to facilitate microscopy. Urease activity still leads to crystalline biofilm formation if urine is used as the medium, but the pH increase is not as extreme as in the multi-well plate biofilm assay and may be maintained within a physiological range. A modification of continuous flow is an artificial bladder A catheter is inserted into the base of the bladder chamber, which may be further connected to tubing and a drainage bag.

The advantage of this bladder model is that it captures several aspects of the urinary tract: However, this model requires specialized equipment and is limited in how many samples can be analyzed at one time. This method may be used to assess aspects of biofilm development that are host-influenced, or require a biotic substrate for the developing biofilm. A drawback is that P.

Animal models are also used to study biofilm development. Stones and clusters form in the mouse ascending UTI model 40 , 42 , 44 , and P.

These models are essential for investigating the interaction of P. Culture medium also greatly affects biofilm development. Using continuous flow coupled with confocal scanning laser microscopy, P. However, in artificial urine , biofilms were flat, lacked channels, and had swarmer cells protruding from the surface Crystalline biofilms will only develop if urine or artificial urine is used.

Catheters in patients will be coated with cellular material and other material not present in artificial urine, and these coatings may further facilitate biofilm formation , Catheters taken directly from patients may be studied to explore polymicrobial biofilms — , or continuous-flow systems , , or static cultures may be inoculated in the lab. Urease activity can give P. The CAUTI mouse model, in which a catheter segment is pushed into and retained in the bladder during the course of the experiment, is also shedding light on P.

Other aspects of mixed-species bacteriuria and infection have been recently reviewed elsewhere 3 , Swarm cell differentiation and the mechanics and regulation of swimming and swarming motility in P. We will therefore sharply focus on the direct contribution of flagella and motility to pathogenesis. A Swarming colony of P. B The swarming migration distance of wild-type strain P19 open circles and a super-swarming rsbA mutant solid circles.

The periodic shift from swarming to consolidation can be seen. C Cartoon and TEM showing differentiated swimmer broth-cultured and swarmer cells. D and F The edge of an advancing swarm colony during consolidation D or swarming F. In contrast to other motile bacteria, all of the flagellar components and chemotaxis proteins of P. Within this region, P. FlaA appears to be the predominant flagellin produced by P. As the flagella produced by P. Flagella are not an absolute requirement for establishment of UTI, as naturally-occurring nonmotile strains and flagellar mutants of P.

However, there is clear evidence for the contribution of flagella to pathogenesis. The importance of flagella to host cell invasion and ascending UTI was directly assessed by disrupting the gene encoding the flagellar filament flaD in a P.

Production of the flagellar filament was found to facilitate invasion of human renal proximal tubular epithelial cells in vitro , partly by allowing the bacterial cells to come into close proximity to the host cells Flagella may also promote internalization through other mechanisms, as the flaD mutant still exhibited reduced internalization compared to the parental strain when the bacteria were centrifuged onto the cell monolayer.

This mutant also exhibited reduced bladder and kidney colonization in the murine model of ascending UTI, while loss of hemolysin alone did not significantly impact pathogenesis. A role for chemotaxis and components of the flagellar apparatus in ascending UTI was also directly indicated by signature-tagged mutagenesis STM studies as a positive regulator of chemotaxis cheW , the flagellar M-ring fliF and flagellar hook protein flgE were all identified as fitness factors for ascending UTI , Transposon insertion in any of these genes resulted in a defect in kidney colonization, while fliF and cheW also affected bladder colonization.

Transposon insertion-site sequencing Tn-Seq similarly indicated a role for chemotaxis and flagella during catheter-associated CAUTI Specifically, a regulator of chemotaxis cheR , the flagellar master regulator flhDC , and a gene involved in regulation of flhDC umoA were identified as fitness factors for kidney colonization, while another gene involved in regulation of flhDC umoB and the flagellar basal-body rod protein flgC were fitness factors for colonization of the kidneys and the catheterized bladder, and the flagellar M-ring fliF and ATP synthase fliI were fitness factors for bladder colonization Thus, the flagellar M-ring has been identified as having a significant contribution to fitness through two separate mutagenesis screens in two distinct models of infection.

It is also noteworthy that flagellar genes are temporally regulated during ascending UTI, being poorly expressed in vivo relative to growth in rich medium at early time points post-inoculation but increasing in expression by 7 dpi, further indicating a role for flagella in pathogenicity Despite numerous studies, the exact contribution of flagella-mediated motility to P.

As non-swarming mutants are only capable of migrating across hydrogel-coated catheters, swarming is likely an important aspect of initial bladder colonization in catheterized individuals. Five cues that can induce swarming in P. A Urine agar is not normally permissive for swarming as the high pH resulting from urease activity inhibits swarming.

A ureC mutant is capable of modest swarming on urine agar, and supplementation with any of five swarming cues to a final concentration of 20 mM dramatically enhances swarming. B Quantitation of swarm colony diameter on urine agar for P. White lines indicate swarm diameter. Error bars represent means and standard deviations for four independent experiments with four replicates each. Statistical significance was determined by comparing the swarm diameter under each condition to the diameter on plain medium for each strain.

Once within the urinary tract, in vitro experiments indicate that swimming motility may facilitate contact with uroepithelial cells, thereby promoting internalization and cytotoxicity Swarm cells in particular have been postulated to contribute to host cell invasion, as these differentiated cells may be capable of invading urothelial cells faster and to a greater extent than vegetative cells Numerous virulence factors are coordinately expressed during swarming, including Zap protease and hemolysin, which may cause swarm cells to be more cytotoxic to the host urothelium 52 , Fig.

The upstream promoter regions of zapA and hpmB , respectively, were fused to a promoterless luxCDABE cassette harbored on a low-copy-number plasmid and transformed into wild-type P. Gene expression was measured as luminescence Lux , which is displayed in false color corresponding to relative Lux intensity, where blue is lowest and red is most intense. An image of the colony growth photographed in natural light is shown below its corresponding Lux image.

Expression of zapA first two rows and hpmB third and fourth row is shown at 4, 8, 12, and 16 h.

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Swimming motility is also thought to contribute to dissemination within the urinary tract, particularly ascension from the bladder to the kidneys and spread between kidneys. For instance, immunization to stimulate production of antibodies that immobilize P. Swarm cells have also been implicated for contributing to kidney colonization and development of pyelonephritis, particularly during long-term infection, as swarm cells have been visualized within the kidney parenchyma 52 , However, elongated swarm cells were rarely observed during four-day infection studies in the uncatheterized murine model of ascending UTI Thus, while flagella clearly contribute to P.

Bacterial pathogens compete with the host for micronutrients. One method the host uses to combat pathogens is sequestration of these nutrients so that the bacteria become starved for these ions. Bacteria that have evolved to colonize humans, therefore, have strategies to scavenge these elements, including iron and zinc , But fc 3: But ic 4: Sa u khi lo? C ac nghifp vu phat sinh trong ky: Y eu cau: Xac djnh so du- cyoi ky cua c ic Tai khoan.

Chiromg 3: Phan anh gia trj tai san hi? Phan anh gia trj ngudn vSn hi? B iitip 3: Hay dinh khoan cac nghiep vu kinh te phat sinh trong ky cua doanh nghiep. H in g hod: B ii tip 5: T ii khoan Trong Qui 2 nam c6 cic nghifp vy kinh te p hit sinh sau: Ye u cau: Related Papers.