Virginia Lockatell

Role of motility in the colonization of uropathogenic Escherichia coli in the urinary tract

ABSTRACT Uropathogenic Escherichia coli (UPEC) causes most uncomplicated urinary tract infections (UTIs) in humans. Flagellum-mediated motility and chemotaxis have been suggested to contribute to virulence by enabling UPEC to escape host immune responses and disperse to new sites within the urinary tract. To evaluate their contribution to virulence, six separate flagellar mutations were constructed in UPEC strain CFT073. The mutants constructed were shown to have four different flagellar phenotypes: fliA and fliC mutants do not produce flagella; the flgM mutant has similar levels of extracellular flagellin as the wild type but exhibits less motility than the wild type; the motAB mutant is nonmotile; and the cheW and cheY mutants are motile but nonchemotactic. Virulence was assessed by transurethral independent challenges and cochallenges of CBA mice with the wild type and each mutant. CFU/ml of urine or CFU/g bladder or kidney was determined 3 days postinoculation for the independent challenges and at 6, 16, 48, 60, and 72 h postinoculation for the cochallenges. While these mutants colonized the urinary tract during independent challenge, each of the mutants was outcompeted by the wild-type strain to various degrees at specific time points during cochallenge. Altogether, these results suggest that flagella and flagellum-mediated motility/chemotaxis may not be absolutely required for virulence but that these traits contribute to the fitness of UPEC and therefore significantly enhance the pathogenesis of UTIs caused by UPEC.

Assessment of Virulence of Uropathogenic Escherichia coli Type 1 Fimbrial Mutants in Which the Invertible Element Is Phase-Locked On or Off

ABSTRACT Type 1 fimbria is a proven virulence factor of uropathogenic Escherichia coli (UPEC), causing urinary tract infections. Expression of the fimbria is regulated at the transcriptional level by a promoter situated on an invertible element, which can exist in one of two different orientations. The orientation of the invertible element that allows the expression of type 1 fimbriae is defined as “on,” and the opposite orientation, in which no transcription occurs, is defined as “off.” During the course of a urinary tract infection, we have observed that the infecting E. coli population alternates between fimbriated and nonfimbriated states, with the fimbriated on orientation peaking at 24 h. We propose that the ability of the invertible element to switch orientations during infection is itself a virulence trait. To test this hypothesis, nucleotide sequence changes were introduced in the left inverted repeat of the invertible element of UPEC pyelonephritis strain CFT073 that locked the invertible elements permanently in either the on or the off orientation. The virulence of these mutants was assessed in the CBA mouse model of ascending urinary tract infection at 4, 24, 48, and 72 h postinoculation (hpi). We conducted independent challenges, in which bladders of mice were inoculated with either a single mutant or the wild type, and cochallenges, in which a mutant and the wild type were inoculated together to allow direct competition in the urinary tract. In both sets of experimental infections, the locked-off mutant was recovered from the urine, bladder, and kidneys in significantly lower numbers than the wild type at 24 hpi (P ≤ 0.05), demonstrating its attenuation. Conversely, the locked-on mutant was recovered in higher numbers than the wild type at 24 hpi (P ≤ 0.05), showing enhanced virulence of this mutant. No significant differences were seen between the mutants and wild type in the urine or the bladder at 48 or 72 hpi. However, the wild type outcompeted the locked-off mutant in the kidneys during the cochallenge experiment at 72 hpi (P = 0.009). Overall, these data suggest that the ability of the invertible element controlling type 1 fimbria expression to phase vary contributes significantly to virulence early (24 hpi) in the course of a urinary tract infection by UPEC and most profoundly influences colonization of the bladder.

In vivo dynamics of type 1 fimbria regulation in uropathogenic Escherichia coli during experimental urinary tract infection

ABSTRACT Escherichia coli is the primary cause of uncomplicated infections of the urinary tract including cystitis. More serious infections, characterized as acute pyelonephritis, can also develop. Type 1 fimbriae of E. coli contribute to virulence in the urinary tract; however, only recently has the expression of the type 1 fimbriae been investigated in vivo using molecular techniques. Transcription of type 1 fimbrial genes is controlled by a promoter that resides on a 314-bp invertible element capable of two orientations. One places the promoter in the ON orientation, allowing for transcription; the other places the promoter in the OFF orientation, preventing transcription. A PCR-based assay was developed to measure the orientation of the invertible element during an experimental urinary tract infection in mice. Using this assay, it was found that the percentage of the population ON in urine samples correlated with the respective CFU per gram of bladder (P = 0.0006) but not with CFU per gram of kidney (P > 0.069). Cystitis isolates present in the urine of mice during the course of infection had a higher percentage of their invertible elements in the ON orientation than did pyelonephritis isolates (85 and 34%, respectively, at 24 h; P < 0.0001). In general, cystitis isolates, unlike pyelonephritis isolates, were more likely to maintain their invertible elements in the ON orientation for the entire period of infection. E. coli cells expressing type 1 fimbriae, expelled in urine, were shown by scanning electron microscopy to be densely packed on the surface of uroepithelial cells. These results suggest that expression of type 1 fimbriae is more critical for cystitis strains than for pyelonephritis strains in the early stages of an infection during bladder colonization.

Macrophages produce TGF-beta-induced (beta-ig-h3) following ingestion of apoptotic cells and regulate MMP14 levels and collagen turnover in fibroblasts.

Phagocytic clearance of apoptotic cells by macrophages is an essential part in the resolution of inflammation. It coincides with activation of repair mechanisms, including accumulation of extracellular matrix. A possible link between clearance of apoptotic debris and accumulation of extracellular matrix has not been investigated. Production of collagen was measured in primary fibroblasts cocultured with macrophages. Ingestion of apoptotic cells by monocyte-derived macrophages led to up-regulation of collagen. Direct contact between macrophages and fibroblasts was not required for collagen up-regulation. Macrophages produced TGF-β following ingestion of apoptotic cells, but the levels of this cytokine were lower than those required for a significant up-regulation of collagen. Simultaneously, the levels of TGF-β-induced (TGFBI), or keratoepithelin/BIGH3, mRNA and protein were increased. In contrast, primary alveolar macrophages stimulated collagen production without exposure to apoptotic cells; there was no further increase in the levels of TGFBI, mRNA or protein, or collagen after ingestion of apoptotic cells. Stimulation of fibroblasts with TGFBI down-regulated MMP14 levels, decreased DNA binding by p53, increased DNA binding by PU.1, and up-regulated collagen protein but not mRNA levels. Overexpression of MMP14 or p53, or small interfering RNA-mediated inhibition of PU.1 led to an increase in MMP14 and a decline in collagen levels, whereas small interfering RNA-mediated inhibition of MMP14 led to elevation of collagen levels. In conclusion, monocyte-derived but not alveolar macrophages produce TGFBI following ingestion of apoptotic cells, leading to the down-regulation of MMP14 levels in fibroblasts through a mechanism involving p53 and PU.1, and to subsequent accumulation of collagen.

Full-Length IL-33 Promotes Inflammation but not Th2 Response In Vivo in an ST2-Independent Fashion

Expression of IL-33 is elevated in patients with pulmonary diseases, and full-length (not proteolytically processed) IL-33 is the predominant form in the lungs in health and disease. To determine whether activation of IL-33 is needed for functional effects, activities of full-length mouse and mature mouse (mm) forms of IL-33 were compared in vivo. Replication-deficient adenoviral constructs were used for gene delivery. Both isoforms caused pulmonary infiltration of lymphocytes and neutrophils, whereas mmIL-33 also caused pulmonary eosinophilia and goblet cell hyperplasia and increased expression of IL-4, IL-5, IL-13, IL-17, MCP-1, and KC. The different effects were not associated with differential release from IL-33–producing cells or by differences in subcellular distributions of IL-33 isoforms. Germline deficiency of the cell surface receptor chain ST2 abrogated the mmIL-33–induced Th2-associated effects (pulmonary eosinophilia, goblet cell hyperplasia, and increased IL-4 and IL-5), yet the lymphocytic infiltration induced by full-length mouse IL-33 or mmIL-33 was not fully abrogated by the absence of ST2. The similar effects of IL-33 isoforms were associated with comparable regulation of gene expression, notably matrix metalloproteinases 3, 10, and 13. Thus, full-length IL-33 is functionally active in vivo in an ST2-independent fashion, and its effects are partially different from those of mature IL-33. The different effects of these isoforms, particularly the pro-Th2 effects of mature IL-33, are due to differential utilization of the IL-33R chain ST2, whereas their similar effects result from regulation of gene expression.

Interleukin-33 Potentiates Bleomycin-Induced Lung Injury

The mechanisms of interstitial lung disease (ILD) remain incompletely understood, although recent observations have suggested an important contribution by IL-33. Substantial elevations in IL-33 expression were found in the lungs of patients with idiopathic pulmonary fibrosis and scleroderma lung disease, as well as in the bleomycin injury mouse model. Most of the observed IL-33 expression was intracellular and intranuclear, suggesting involvement of the full-length (fl) protein, but not of the proteolytically processed mature IL-33 cytokine. The effects of flIL-33 on mouse lungs were assessed independently and in combination with bleomycin injury, using recombinant adenovirus-mediated gene delivery. Bleomycin-induced changes were not affected by gene deficiency of the IL-33 receptor T1/ST2. Combined flIL-33 expression and bleomycin injury exerted a synergistic effect on pulmonary lymphocyte and collagen accumulation, which could be explained by synergistic regulation of the cytokines transforming growth factor-β, IL-6, monocyte chemotactic protein-1, macrophage inflammatory protein\x{2013}1α, and tumor necrosis factor-α. By contrast, no increase in the levels of the Th2 cytokines IL-4, IL-5, or IL-13 was evident. Moreover, flIL-33 was found to increase the expression of several heat shock proteins (HSPs) significantly, and in particular HSP70, which is known to be associated with ILD. Thus, flIL-33 is a synergistic proinflammatory and profibrotic regulator that acts by stimulating the expression of several non-Th2 cytokines, and activates the expression of HSP70.

Regulation of pulmonary inflammation and fibrosis through expression of integrins αVβ3 and αVβ5 on pulmonary T lymphocytes

OBJECTIVE Pulmonary diseases associated with fibrosis, including scleroderma lung disease, are characterized by the accumulation of T cells in the lungs. These cells are thought to facilitate lung fibrosis, but the exact mechanisms of their profibrotic action are not clear. Several alphaV-containing integrins, including alphaVbeta3 and alphaVbeta5, have been shown to directly activate transforming growth factor beta (TGFbeta) and promote collagen accumulation. The aim of this study was to investigate whether pulmonary T cells express profibrotic integrins and regulate collagen accumulation. METHODS Expression of integrins was assessed by immunohistochemical analysis of lung tissue, by flow cytometry using bronchoalveolar lavage fluid from patients with systemic sclerosis (SSc), and in a CCL18 overexpression animal model of pulmonary T cell infiltration. Experiments in cell cultures were performed to determine whether integrin-expressing T cells are profibrotic in cocultures with pulmonary fibroblasts and, if so, through what possible mechanism. RESULTS Lymphocytes and integrin-positive cells were present in the lungs, and pulmonary T cells expressed integrins alphaVbeta3 and alphaVbeta5 in patients with SSc and in the animal model. Systemic administration of neutralizing anti-integrin alphaV antibody or a genetic deficiency of integrin beta3 in the CCL18 overexpression model significantly attenuated CCL18-driven pulmonary lymphocytic infiltration and collagen accumulation. Jurkat T cells overexpressing integrin alphaVbeta3 or integrin alphaVbeta5 in cocultures with primary pulmonary fibroblasts stimulated collagen accumulation and Smad2 nuclear translocation. Neutralizing anti-TGFbeta antibody attenuated the profibrotic effect of integrin-expressing T cells. CONCLUSION Pulmonary infiltrating T lymphocytes may express integrins alphaVbeta3 and alphaVbeta5 that are necessary for lymphocytic infiltration and T cell-associated TGFbeta activation and collagen accumulation.

Mannose-Resistant Proteus-Like Fimbriae Are Produced by Most Proteus mirabilis Strains Infecting the Urinary Tract, Dictate the In Vivo Localization of Bacteria, and Contribute to Biofilm Formation

ABSTRACT Proteus mirabilis, an etiologic agent of complicated urinary tract infections, expresses mannose-resistant Proteus-like (MR/P) fimbriae whose expression is phase variable. Here we examine the role of these fimbriae in biofilm formation and colonization of the urinary tract. The majority of wild-type P. mirabilis cells in transurethrally infected mice produced MR/P fimbriae. Mutants that were phase-locked for either constitutive expression (MR/P ON) or the inability to express MR/P fimbriae (MR/P OFF) were phenotypically distinct and swarmed at different rates. The number of P. mirabilis cells adhering to bladder tissue did not appear to be affected by MR/P fimbriation. However, the pattern of adherence to the bladder surface was strikingly different. MR/P OFF colonized the lamina propria underlying exfoliated uroepithelium, while MR/P ON colonized the luminal surfaces of bladder umbrella cells and not the exfoliated regions. Wild-type P. mirabilis was usually found colonizing intact uroepithelium, but it occasionally adhered to exfoliated areas. MR/P ON formed significantly more biofilm than either P. mirabilis HI4320 (P = 0.03) or MR/P OFF (P = 0.05). MR/P OFF was able to form a biofilm similar to that of the wild type. MR/P ON formed a three-dimensional biofilm structure as early as 18 h after the initiation of the biofilm, while MR/P OFF and the wild type did not. After 7 days, however, P. mirabilis HI4320 formed a 65-μm-thick biofilm, while the thickest MR/P ON and MR/P OFF biofilms were only 12 μm thick. We concluded that MR/P fimbriae are expressed by most P. mirabilis cells infecting the urinary tract, dictate the localization of bacteria in the bladder, and contribute to biofilm formation.

Alternatively spliced variants of interleukin-4 promote inflammation differentially

IL‐4δ2 is a natural splice variant of IL‐4 that lacks the region encoded by the second exon. Numerous reports have suggested that the expression levels of IL‐4δ2 change in various diseases, especially those with pulmonary involvement, but the in vivo effects of this splice variant have never been studied. Replication‐deficient, AdV‐mediated gene delivery of mIL‐4δ2 to mouse lungs in vivo was used, and the effects compared with similar adenoviral delivery of mIL‐4 or with infection with a noncoding NULL viral construct. Overexpression of IL‐4δ2 or IL‐4 caused pulmonary infiltration by T and B lymphocytes, whereas in contrast to IL‐4, IL‐4δ2 did not induce eosinophilia or goblet cell hyperplasia. Microarray analysis of global gene expression revealed that IL‐4δ2 and IL‐4 had differential effects on gene expression. These splice variants also differentially regulated pulmonary levels of the cytokines TNF‐α, eotaxin, IL‐1α, IFN‐γ, and MCP‐1, whereas both tended to increase total lung collagen modestly. Pulmonary infiltration by lymphocytes in response to overexpression of IL‐4δ2 was attenuated but not abrogated completely by germline deficiency of IL‐4Rα or STAT6, whereas deficiency of endogenous IL‐4 had no effect. Thus, IL‐4δ2 promotes lymphocytic inflammation in vivo (although differentially from IL‐4, in part), and the effects of IL‐4δ2 are not mediated by endogenous IL‐4. Differential targeting of IL‐4δ2 and IL‐4 may therefore be considered in developing future therapeutic agents.

Splice isoforms of human interleukin-4 are functionally active in mice in vivo

Interleukin‐4 (IL‐4) acts on cultured cells in a species‐specific fashion, although several reports have suggested that human (h) IL‐4 may be functionally active in rodents in vivo. The latter finding, if true, would not only offer possibilities for pre‐clinical testing of novel hIL‐4‐targeting therapies in animals, but also suggests new opportunities for mechanistic studies of IL‐4 and its receptors. Conventional IL‐4 is encoded by four exons, whereas its poorly studied alternatively spliced isoform is encoded by exons 1, 3 and 4 (IL‐4δ2). Replication‐deficient adenovirus‐mediated gene delivery of hIL‐4 isoforms (hIL‐4 or hIL‐4δ2) to mouse lungs caused similar pulmonary infiltration of T and B lymphocytes, but not eosinophils. There were significant differences in the changes of pulmonary cytokine milieu induced by hIL‐4 compared with hIL‐4δ2, with hIL‐4δ2 inducing higher levels of pro‐inflammatory (tumour necrosis factor‐α, IL‐1, and monocyte chemotactic protein‐1) and T helper type 1 (IL‐12 and interferon‐γ) cytokines. There was no elevation in endogenous mouse (m) IL‐4 or mIL‐4δ2 mRNAs, and germ‐line deficiency of mIL‐4 did not affect the degree of pulmonary infiltration. When combined with an ovalbumin model of asthma, hIL‐4δ2 stimulated a greater accumulation of lymphocytes than did hIL‐4. Pulmonary infiltration of lymphocytes induced by expression of hIL‐4 or hIL‐4δ2 was attenuated, but not completely abrogated, by germ‐line deficiency of mIL‐4Rα or murine signal transducer and activator of transcription 6, suggesting that these signalling molecules mediate the in vivo effects of hIL‐4 isoforms in mice. These findings suggest that splice isoforms of human IL‐4 are functionally active in vivo in mice, and partially share the effects of the corresponding species‐specific isoforms.