Mutation in uvrY or barA decreases the virulence in a mouse UTI model
The UPEC can cause cystitis or pyelonephritis. The UPEC originate from distal gut, colonize the vagina and/or ascend the urinary tract to the bladder via the urethra 
. The UPEC have the ability to invade the urinary tract and develop biofilms 
. To investigate the effect of the uvrY or barA
mutant on bacterial pathogenesis, we developed ascending urinary tract infection via transurethral catheterization in a mouse model. To evaluate the virulence, bacterial load was determined in various tissues and urine. Mutation in either barA
exhibited reduced colonization as compared to the wild-type in mouse ascending UTI model (). The barA
mutant bacteria colonized less efficiently in both bladder and kidneys, while uvrY
mutant bacteria colonized less efficiently in the bladder of the mice when compared to the wild-type. The bacterial loads of barA
mutant colonized in bladder and kidneys were significantly reduced by two and one log10 CFU/gram of tissue as compared to wild-type (*P<0.01). The uvrY
mutant demonstrated significant reduction of bacterial loads in bladder by one log10
CFU/gram of tissue when compared to that of wild-type (*P<0.01, ). Our result showed that the load of mutant was slightly reduced in the urine by approximately one log10
CFU/ml, which may further explain the poor colonization of the bladder and kidneys. Using E. coli
DS17 it has been shown that the uvrY
mutant has a lesser fitness for survival in urine in a primate infection model 
. Here, we have showed the extent of colonization of both barA
mutant in the tissues of the urogenital system.
Mutation in barA or uvrY reduces virulence in murine ascending UTI.
Mutation in barA or uvrY affects hemolysin secretion
Differential expression of virulence factors in UPEC might be an important factor for colonizing either in the bladder causing cystitis or in kidney resulting in pyelonephritis. Since the barA
mutant showed decreased colonization in mouse UTI model, chicken embryo killing and reduced cytotoxicity to cultured HK-2 cells, we further compared the secreted protein profiles in the culture supernatants from the various mutants and wild-type. By Coomassie blue staining, we identified a unique band of approximately 110 kDa that was reduced in its expression level in either barA
mutant. By mass spectrometric analysis, this band was further identified as Hemolysin (HlyA) 
(). The exotoxin HlyA is one of the virulence factors associated with pathogenesis; the production of hemolysin protein contributes to the virulence of extra-intestinal pathogenic E. coli
. E. coli
bacterial suspensions containing hemolysin treated onto cultured human kidney proximal tubular epithelial cells resulted in highly elevated cytotoxicity; transurethral challenge in CBA mice resulted in pyelonephritis 
. The HlyA was down-regulated in the uvrY
mutant (, lane 6) compared to wild-type (, lane 3). Furthermore, we investigated the hemolytic function of the secreted hemolysin protein from bacterial culture supernatant by using sheep erythrocytes at 30.5°C for 7 hr. The hemolytic activity of barA
mutants was significantly reduced when compared to wild-type (***, p≤0.001). There was more than 5-fold or 7-fold decrease in hemolytic activity in the mutant barA
or mutant uvrY
respectively (). Functional complementation of both mutant genes by plasmids restored the hemolytic activity similar to the level of wild-type (). The decreased activity of the exotoxin HlyA may also contribute to the reduction of the virulence in barA
mutant bacteria. Taken together, the hemolytic activities in supernatants from the barA
mutants might contribute and well correlated to the reduced cytotoxic effects against human kidney HK-2 cells and decreased chicken embryo mortality.
Protein profiles of wild type and mutant bacteria supernatant.
Mutation in barA or uvrY decrease and csrA increase hemolysis of sheep erythrocytes.
Deletion of uvrY affects LPS profile
is a potent endotoxin responsible for gram negative septicemia 
and also responsible for the production of a variety of proinflammatory cytokines followed by septic shock and disseminated intravascular coagulation of the infected animals 
. Next, we investigated the role of various mutants in UPEC lipopolysaccharide biosynthesis. As shown in the , LPS was isolated from equal number of cells and resolved in reducing 12% SDS-PAGE. The LPS profile of the uvrY
mutant exhibited visible differences in the core and O-antigen compared to that of LPS from wild-type. Mutation in uvrY
(, lane 4), caused differences in both migration pattern of O-antigen bands and quantity of the LPS as compared to the LPS from wild-type (, lane 1). Some bands of the O-antigen were missing and few other bands were less prominent when compared LPS in WT CFT073. Complementation of the uvrY
mutant (, lane 5) in trans
restored the WT phenotype of LPS. In csrA
mutant LPS, one high molecular weight O-antigen band became less prominent and few low molecular weight O-antigen bands showed more prominence (indicated by arrows in , lane 6). The csrA/
complemented strain had reduced LPS expression (, lane 7). Quantification of the LPS from equal number of E. coli
) by densitometry, titrated the amount of LPS from uvrY
(, lane 4, 723 ng/µl) mutant strain was partially reduced when compared to the wild-type (, lane 1, 853 ng/µl) and the complementation of uvrY
mutant restored the WT phenotype (, lane 5, 1065 ng/µl). One possible mechanism is that mutation of uvrY
may modulate the rfa
gene cluster responsible for LPS biosynthesis resulting in differential expression of LPS. Loss of LPS or changes in LPS profile may also lead to reduction in colonization and invasiveness. Also rough strains do not persist in vivo as efficiently as the smooth strains. This might help in explaining uvrY
mutant is not so invasive or have low level of colonization in mouse UTI model.
Lipopolysaccharide profiles of wild type and mutant bacteria.
Mutation in uvrY down-regulates inflammatory cytokines
LPS is an integral part of outer membrane of UPEC. Either bacterial infection or LPS treatment triggers cytokine production in mucosal tissue 
. The epithelial cells lining urinary tract play a major role in host pathogen interaction by secreting various cytokines in response to infections. First, UPEC invades and colonizes in uroepithelial cells, which triggers the release of proinflammatory cytokines. It has been shown that the uroepithelial cell lines secrete interleukin-6 (IL-6) and chemokine IL-8 when stimulated by UPEC 
. During gram negative septicemia, the proinflammatory cytokines like tumor necrosis factor α (TNF-α) and IL-6 and chemokine IL-8 activate the inflammatory cascade and trigger the systemic infection 
. Here, we further investigated the cytokine responses of human uroepithelial cell line SV-HUC-1 cells to infection with either whole bacterial cells or treatment with purified LPS alone by quantitative RT-PCR analysis (). Both whole bacterial cells and LPS treatment stimulated the mRNA expressions of TNF-α, ΙL-6 and IL-8. Their expression levels were higher in SV-HUC-1 cells infected with whole bacterial cells than in cells treated with LPS alone. Possible explanation for this higher expression of TNF-α, ΙL-6 and IL-8 from stimulation with whole bacterial cells may be due to surface expression of virulence factors like Type 1 or P fimbriae, capsule, outer membrane proteins or O-specific antigen and toxins including hemolysin and LPS. The infection with WT bacteria and its LPS treatment had the highest expression of all three cytokines. These results support the previous findings 
while infection with uvrY
mutant bacteria down-regulated TNF-α, IL-6 and IL-8 by ~4, 5 and 9-folds compared to the WT bacteria (**, p≤0.01) (). The cells treated with purified LPS from uvrY
mutant also down-regulates the cytokines TNF-α and IL-6 by ~3-folds compared to the WT LPS (*, p≤0.05). The uvrY
complemented strain restored the effects on the level of cytokine production similar to that of wild-type (). The difference in the LPS profile pattern () may contribute to the lowered expressions of these cytokines. These factors might also contribute to the reduction in virulence of the uvrY
Mutation in uvrY gene down regulates inflammatory cytokine expression.
Majority of UTI were caused by UPEC 
. Our results with murine UTI showed the important roles of BarA-UvrY TCS in the virulence of UPEC. Mutation with barA
reduced the invasion and colonization in bladder and kidneys, decreased chicken embryo killing, and reduced cytotoxicity to HK-2 cells (, & ; ). There was 1–2 log reduction in bacterial colonization and 58–67% reduction in chicken embryo killing, suggesting that the pathogenicity and virulence factors of UPEC CFT073 were controlled by various virulence determinants. Hence, mutation in BarA-UvrY TCS was less efficient and various other TCS and signaling pathways may also contribute to the virulence of UPEC CFT073. Between the mutations in BarA-UvrY TCS, effect of uvrY
mutation is more pronounced and less virulent as compared to mutation in barA
, a membrane sensor protein. This conclusion is strongly supported by reduction of other virulence determinants like LPS, hemolysin and proinflammatory cytokines and chemokine (& , &).
It has been shown that CsrA is a RNA binding protein. More interestingly, mutation in csrA
increased the virulence of UPEC CFT073. This conclusion is supported by several evidences. First, deletion of csrA
in strain CFT073 increased the virulence to 75% (3 out of 12 survived) in a chicken embryo lethality assay (). The increased virulence was reduced to 50% in a complemented csrA/
strain. This result is in agreement with a previous study that mutation of csrA
in E. coli
K-12 enhanced biofilm formation by regulating intracellular glycogen biosynthesis and catabolism and over expression of csrA
repressed biofilm formation 
. These results suggest that csrA
gene represses certain virulence factors; CsrA may exhibit distinct mechanism from BarA-UvrY TCS in modulating virulence factors. Second, deletion of csrA
gene which controls carbon metabolism and flagellum biosynthesis 
resulted in unaltered invasiveness in ureter (SV-HUC-1) cells while the csrA
complemented mutant strain reduced the invasiveness by one log (~10-fold) in SV-HUC-1 uroepithelial cells ().
The differences exhibited by the invasion ability of csrA mutant in ureter (SV-HUC-1) may be due to the growth rate of the csrA mutant or owing to the changes in the expression of virulence factor during invasion. Indeed, mutations of csrA gene affect the growth of the bacteria and these mutants tend to grow slowly. Third, csrA mutant was more cytotoxic () compared to its wild-type (), where only 13% of treated HK-2 cells () were alive and mutation in csrA gene resulted in moderate increase in hemolytic activity and its complementation in plasmid decreased the hemolytic activity, indicating that CsrA represses the hemolysin expression (). Fourth, the csrA mutant had the highest concentration of LPS (, lane 6, 2080 ng/µl) and csrA/p-csrA complemented strain led to reduction in the level of LPS expression (, lane 7, 643 ng/µl). Interestingly, even though the csrA mutant had the highest concentration of LPS (, lane 6, 2080 ng/µl) from equal number of bacterial cells, SV-HUC-1 cells treated with equal amount of LPS (10 ng/µl) did not produce the cytokine levels like that of wild-type (). Taken together, our assay demonstrated that increased virulence determinants, like hemolysin activity and LPS, may contribute to the enhanced virulence in csrA mutation and over-expression of csrA suppresses the bacterial virulence. However, the increase in virulence seen with the csrA mutant in these in vitro experiments needs to be further verified by in vivo animal experiments.
In spite of this, our in vitro
experimental results are in agreement with the previous study that csrA represses pgaABCD
transcript involved in the synthesis of polysaccharide adhesion, thereby repressing biofilm formation in E. coli
, but different from another study that mutation of csrA
gene in Salmonella enterica
serovar Typhimurium reduces the invasion of HEp-2 epithelial cells and expression of Salmonella
pathogenicity island 1 (SPI1) invasion genes 
. However, over-expression of csrA in trans
also suppress the expression of SPI1 invasion genes 
. Also, in Legionella pneumophila
, over-expression of CsrA suppress virulence associated traits and mutation of csrA
gene result in increased virulence associated gene letE
, stationary-phase sigma factor, RpoS and enhance flaA
genes resulting in premature flagellation 
. These studies, including ours, demonstrated that CsrA acts as both repressor and enhancer of invasion genes and its expression could be tightly regulated. The future study can be directed to examine the expression level of CsrA protein in the uvrY
mutant and establish a functional relationship between BarA-UvrY TCS and CsrA in regulating the expression of virulence genes either positively or negatively.
In summary, our work further delineates the role of BarA-UvrY TCS in regulating various virulence factors. In UPEC, the Cpx two-component signal transduction system, CpxA/CpxR controls P pilus biosynthesis and regulates phase variation of pap
and other virulence factors 
. It is also reported that pyelonephritogenic E. coli
strains are more cytotoxic to cultured human renal tubular epithelial cells and the cytotoxin hemolysin contributes to this potent virulence as well 
. A previous study showed that disruption of the BarA-UvrY TCS reduced the fitness of the uvrY
mutant in a monkey cystitis model 
. Similarly, in our assay, mutation in uvrY
reduces the cytotoxicity to the cultured human kidney (HK-2 cells) and hemolysin production ( & , & ). Previously our microarray data showed and we also hypothesize that BarA-UvrY two-component system may regulate the expression of pap
gene cluster encoding P pilus, fimbrial genes encoding Type 1 fimbriae, outer membrane protein such as ompC
or LPS biosynthesis genes like rfa
gene cluster, hly
locus encoding hemolysin synthesis and secretion, which may directly or indirectly contribute to the virulence of the UPEC 
. Further investigation is needed to understand the role of these genes in the pathogenesis of UPEC in the UTI. Taken together, our results show that BarA-UvrY TCS regulates various virulence determinants contributing to the pathogenicity of the UPEC CFT073.