Initial events in the colonization of the urinary tract by UPEC remain to be characterized in detail. We hypothesize that navigation to and in the urinary tract via chemotaxis are among the first steps to aid UPEC colonization of the urethra and bladder. As previously shown, uropathogenic E. coli
CFT073 is attracted to undiluted human urine, demonstrating that the attraction of CFT073 to urine may aid the bacteria in gaining access to the urinary tract 
. In support of this hypothesis, other bacterial species use chemotaxis to direct colonization of specific regions of the digestive tract (e.g. Vibrio cholerae
to the deep crypts of the small intestine and Campylobacter jejuni
to the mucus of the gastric epithelium) 
. Additionally, the plant pathogen Ralstonia solanacearum
uses aerotaxis to quickly find the roots of its host plant to initiate colonization 
. Multiple species of bacteria use chemotaxis to direct colonization to their preferred niches, highlighting chemotaxis as an important colonization factor during pathogenesis.
We sought to determine which chemoreceptor(s) is responsible for the attraction of CFT073 to urine. CFT073 deletion mutants missing one of the three chemoreceptors (Δtsr
, or Δaer
) were subsequently tested for chemotaxis toward human urine. Loss of any one chemoreceptor did not result in significant loss of chemotaxis toward urine, indicating that there were redundant ligands shared between the chemoreceptors and/or that there were multiple strong attractants in urine that are sensed by different chemoreceptors. When both tsr
were absent, the positive response to urine was almost abolished, indicating that these two chemoreceptors are mainly responsible for chemotaxis toward urine. The loss of aer
did not change the response to urine to a significant degree. One explanation for the loss of a response toward urine when only aer
is present may be that the small number of Aer receptors, which is markedly lower than Tsr and Tar levels 
, is simply too small to direct an efficient chemotactic response without the aid of Tsr and Tar to form the receptor clusters 
. Therefore, urine could still stimulate a response through Aer, but the resulting signal would be too small to effect a chemotactic response that could be detected by the capillary assay. Aer could also be important for indirectly sensing oxygen within the urinary tract, but aerotaxis was not tested in this study.
The results obtained in this study with E. coli CFT073 mostly concur with the results obtained previously with E. coli K-12. Just as with K-12, CFT073 is attracted to a variety of L-amino acids including alanine, asparagine, aspartate, cysteine, glutamate, glycine and serine. Methionine, threonine and histidine also stimulated accumulation of the bacteria in the capillary tubes, but these responses were not statistically significant. The apparent dose response to L-methionine indicates that this amino acid may be a weak attractant for CFT073 despite the lack of statistical significance. The responses obtained in this report were also slightly lower than in Adler’s original report, a difference that might be accounted for by growth in urine instead of MOPS glycerol and some slight differences in the purity of the attractants used. The increased response of CFT073 toward lower concentrations of L-serine and L-aspartate demonstrates that an optimum concentration of each attractant exists that will stimulate chemotaxis. Concentrations over that optimum may saturate the receptors and decrease chemotactic behavior. Therefore, the optimum concentration of attractants in the urine of an individual may be necessary to maximize the ability of the bacteria to ascend the urinary tract. The optimum concentrations for L-aspartate and L-serine are below the highest reported concentrations of each amino acid in human urine. This suggests that concentrated urine with the highest levels of L-aspartate and L-serine may dampen the chemotactic response of CFT073.
The chemoreceptor mutant experiments to determine whether Tsr or Tar mediates the response to each L-amino acid did agree with previous reports 
. The chemoreceptor deletion mutants also illustrate that the chemoreceptors are needed to see the response to each attractant, indicating that the accumulation of bacteria detected in the capillary tubes is due to a true chemotactic response. Accumulation for a few of the attractants was still detected after the deletion of tsr
, a result that could indicate that both Tsr and Tar are capable of sensing the particular attractant or that the particular attractant stimulates chemokinesis as well as chemotaxis. Additionally, this study identified Aer as a partial mediator of chemotaxis toward L-alanine and L-cysteine in E. coli
In addition to the response to L-amino acids, this study also identified several D-amino acids and two non-amino acid components of urine that produced accumulation of E. coli CFT073 above buffer within their physiological ranges. However, the results for these components were not statistically significant above buffer and may not be truly capable of eliciting a response through the chemoreceptors. These components include D-alanine, D-phenylalanine, D-proline, D-serine, D-threonine, D-tyrosine, glucose and caffeine with D-serine exhibiting the strongest response. The other D-amino acids on this list as well as glucose and caffeine weakly responded even at increased concentrations, although D-asparagine generated a response that surpassed that of D-serine when tested at a concentration that far exceeded its reported concentration range in urine. Since concentrations of each amino acid can vary widely from person to person, the reported range of D-asparagine in human urine is by no means absolute. Therefore, D-asparagine may act as an attractant in urine for UPEC when its concentration is sufficiently high. Capillary assays using the mutant strains with allelic repair of the deleted chemoreceptor gene showed that only the responses toward D-alanine, D-asparagine and D-serine could be returned to wild-type levels. This suggests that, from the above group, only these three D-amino acids may elicit a true response through the chemoreceptors.
Our results also indicate that loss of any two of the chemoreceptors does not affect colonization of the urinary tract in the mouse model. However, the direct inoculation of the bladder and potential reflux of bacteria into the kidneys of the mice immediately after inoculation prevents any conclusions that can be made about the necessity of chemotaxis for ascension of the urethra and ureters and could mask any potential colonization defects. A better model for ascension of the urinary tract is needed to address this issue. Additionally, the chemotaxis system could be important for not just ascension of the urinary tract but penetration of the mucus layer to achieve close contact with the bladder epithelium 
. Without the ability to switch rotation of the flagella and the direction of net movement, the bacteria could become stuck in the mucus layer leading to eventual expulsion of the bacteria from the urinary tract by micturition. One final curious observation is that, while the CFT073Δtsr
mutant was attenuated for chemotaxis toward urine in vitro
, this strain performed like wild-type CFT073 in the mouse model. Although Aer protein levels appear insufficient to produce a detectable response in the capillary assay, they may be sufficient to produce a chemotactic response or switching of the flagellar motor that keeps the bacteria competitive during infection. Reflux during inoculation may also conceal any defects that may be observed in vivo
using the CFT073Δtsr
This study has shown that amino acids, possibly D-forms as well as L-forms, act as attractants for E. coli CFT073 within their physiological concentrations in urine and that Tsr and Tar are primarily responsible for sensing these attractants. Loss of any two chemoreceptors did not result in a fitness defect in vivo, although the current mouse model may be a poor tool to evaluate the contribution of each chemoreceptor in the infectious process. Despite this limitation, a possible set of ascension steps in which chemotaxis may play a role can be imagined. Ascension aided by chemotaxis from the relatively nutrient poor region of the urethral opening to the bladder may be stimulated by exposure to urine during voiding of the bladder. The short female urethra may also allow the bacteria to gain access to the bladder as a result of physical trauma to the urethral area. Ascension from the bladder to the kidneys may occur after increasing numbers of growing bacteria in the bladder consume most of the available nutrients, thus creating a gradient from the bladder up the ureters. This gradient may then stimulate chemotaxis and enhanced movement up the ureters to the kidneys. The ability to navigate the urinary tract may result in more efficient colonization, lessening the possibility that the bacteria would be dispelled by urine before being able to colonize the bladder. An improved method for monitoring early events in the colonization of the urinary tract are needed to determine if chemotaxis plays a part in the ascension of the urethra and ureters to colonize the urinary tract.