Human Insulin Modulation of Escherichia coli Adherence and Chemotaxis

Escherichia coli exhibited increased hydrophobicity and mannose-resistant epithelial cell adherence after growth in the presence of human insulin (2 μU mL ̄ or 200 μUmL ̄ insulin, respectively) with glucose (100 mg dL ̄). Capsule production and hemagglutination were unaffected by insulin and glucose. Chemotactic attraction to glucose as compared to insulin or glucose alone was enhanced by the presence of insulin. Insulin alone (200 μU mL ̄) was a chemorepellent and inhibited flagellar tethering to glass. These findings indicate that human insulin can modulate E. coli’s expression of factors associated with pathogenesis in a manner that is modifiable by the presence of glucose.


INTRODUCTION
Escherichia coli is the most common etiologic agent of bacteremia, urinary tract infections (UTI) and acute pyelonephritis [1] . Any organism which causes systemic infections is exposed to a variety of host factors including hormones. The most prevalent and pervasive of these hormones is insulin. Previous studies determined that E. coli alters its growth kinetics in response to human insulin [2] . This insulin mediated change in doubling time is glucose dependent and reminiscent of quorum sensing, i.e., population size, dependent. However, other than affecting growth rates, insulin's effect on E. coli phenotype is unknown. The focus of this study was to determine the effect human insulin has on expression of factors reported to be associated with E. coli's ability to colonize, i.e., adherence-related colonization factors and motility.
Quorum sensing: Autoinducers I and II were measured as described by Surette et al. [3] with Vibrio harveyi strains generously provided by Bonnie Bassler (Princeton University, NJ). Positive controls were filtrates from Vibrio harveyi BB152 and BB120 grown in medium with (200 µU mL¯1) and without insulin and/or glucose (0.2 gm dL¯1). E. coli were grown in Mueller-Hinton broth containing insulin (200 µU mL¯1) and/or 0.2 gm dL¯1 glucose. Mid-log, early stationary and late stationary growth phase (24 hr) cells were pelleted (12,000 X g, 10 min, RT) and the supernatant filter sterilized then stored (-70°C) until use. V. harveyi BB170 and BB886 (O/N culture, 30°C, 1:5 X 10 3 dilutions) luminescence (Coleman luminometer) was measured hourly after exposure to cell filtrate. Negative controls contained sterile medium.
The production of acidic polysaccharide capsule was determined essentially as described for surface hydrophobicity [4] except that bacteria were stained with Alcian blue (0.3 gm dL¯1 PBS, 30 min, room temperature; EIA reader, Dynatech Laboratories, Inc., Chantilly, Virginia; 450 nm).
Epithelial cell adherence: Human buccal and uroepithelial cells were obtained and stored until use as previously described [5][6][7] . The adherence assay was performed as described by Leffler et al. [8] . E. coli (0.05, Abs 540nm ) grown in Mueller-Hinton broth alone (growth control) or with insulin (200 µU mL¯1) and/or 0.2 gm dL¯1 glucose (St. Louis, MO) were washed and suspended (10 8 CFU mL¯1) in PBS with and without 2 gm dL¯1 mannose. One ml of bacterial suspension was mixed and incubated (30 min, 37ºC, 200 rpm) with an equal volume of uroepithelial or buccal cells (10 5 cells mL¯1). The negative control was epithelial cells with 1 ml of PBS alone. After incubation, unattached bacteria were removed by centrifugation (250 X g, 10 min; 4X). The suspension was then dried, fixed and stained with crystal violet. The number of E. coli adherent to 50 epithelial cells assay -1 was counted.
Hemagglutination: Defibrinated sheep erythrocytes (Remel, Lenexa, KS) were washed twice in PBS and suspended to a final concentration of 0.5 cells dL¯1. E. coli (50 µL¯1, 10 10 cells mL¯1 PBS with and without 2 gm dL¯1 mannose) grown as described above for epithelial cell adherence were mixed with an equal volume of erythrocytes, then incubated at room temperature for 2-4 hours before reading [9] .
Chemotaxis: Chemotaxis was assessed essentially as described by Adler [10,11] with the geotactic effects controlled by placement of capillary tubes in 5ml beakers containing 0.5 mL¯1 of bacteria suspension instead of U-tubes.
Mid-logarithmic growth phase cells (37 0 C, shaken; OD 590 of 0.4) were harvested by centrifugation at 6,000 3 X g for 3 min. The pellet was washed twice in chemotaxis medium (10 mM K 2 HPO 4 , 0.1 mM EDTA) pH 7.0 then adjusted to a final concentration of 7 x 10 6 CFU mL¯1.
Analysis of free-swimming behavior: Swimming behavior of cells suspended in PYNB (OD 590 of 0.1) with and without insulin and/or glucose, as described above, was assessed by bright-field microscopy (Petroff-Hauser counting chamber) and the extent of translocational movement and tail-spinning (flagellar tethering) determined.
Statistical evaluation: All assays were done in triplicate and repeated at least twice. Whenever possible, experiments were coded and performed in a blinded fashion. Analysis of variance was used to determine differences between experimental conditions with P < 0.05 considered significant. If statistical significance was found, a Tukey-Kramer post-hoc analysis was applied (Instat, GraphPad Software, San Diego, CA).

RESULTS AND DISCUSSION
Human insulin in the presence of glucose affects expression of colonization factors. Insulin with glucose significantly (P < 0.05) enhanced adherence, biofilm formation and chemotactic response to glucose as compared to medium/buffer alone or medium/buffer with glucose ( Table 1). The adherence to epithelial cells or erythrocytes was not affected by the presence of mannose. Microscopically, mid-logarithmic E. coli cells grown in insulin and glucose (200 µU mL¯1 insulin; 200 mg dL¯1 glucose) were elongated, appeared tethered by their flagella to glass and exhibited clockwise and counterclockwise rotation [12][13][14] . With the exception of chemorepulsion, insulin in the absence of glucose exhibited no other measurable effect on the virulence factors examined. Similar to the effects of insulin and glucose on growth rate [2] , the effects of insulin and glucose on virulence factor expression were concentration specific.
Capsule production and hemagglutination were unaffected by the presence of insulin with or without glucose at all concentrations and combinations tested.
Some of the insulin effects measured may be due directly, or indirectly to the presence of homoserine lactone (AI-1) or cyclic borate diester (AI-2) which are autoinducers of quorum sensing and altered virulence factor production [3,15,16] . Addition of cell-free culture fluid of E. coli strains to either V. harveyi reporter strain BB170 (autoinducer-2) or BB 886 (autoinducer-1), showed no light production under conditions containing glucose and/or insulin indicating that under these growth conditions acyl-homoserine lactone autoinducers are not produced by E. coli.
Prior studies show that E. coli K12's microbial insulin has chemical, immunological and biological properties similar to that of mammalian, worm and protozoan insulin, although basic local alignment search tool (BLAST) analysis comparing human insulin and the E. coli genome indicated a lack of significant alignments which is not entirely surprising since human insulin is post-translationally processed [17][18][19] .
In addition to affecting growth characteristics [2] , insulin with glucose also enhances adherence to epithelial cells, which is considered to be the first step in colonization of host mucosal surfaces. It is well documented that E. coli grown in medium with glucose exhibits an increased ability to adhere to uroepithelial cells [20] . Our findings indicate that insulin augments the effect of glucose thereby boosting epithelial cell adherence. This adherence may be mediated by a variety of adhesive factors [20][21][22][23][24][25][26][27] which are affected by insulin including fimbriae (P-pilus, because of mannose-resistance) and/or hydrophobic interactions. Microscopic examination of the motile cells also showed a tethering of cells to glass via their flagella in the presence of insulin and glucose but not insulin alone. Increased adherence to glass, a negatively charged surface, is reported to be indicative of ionic interactions and increased bacteria cell surface positive charge [28] . Increased positive surface charge has been correlated with an increased ability to interact with mammalian cell surfaces which carry a negative charge [29] . The tethering and rotational movement may also be reflective of localized changes in flagella composition resulting in electrostatic changes.
However, while flagella have been reported to mediate adherence in enteropathogenic E. coli [30] its role in E. coli K12 cellular adherence has not been established. Flagella can also play another role in uropathogenesis. For Proteus mirabilis motility has been shown to contribute to virulence in ascending UTIs [31] . Whether motility and directional translocation play a similar role in the establishment of E. coli urinary infections remains to be determined. It is interesting that in the absence of glucose, an energy source, insulin acts as a chemo-repellent. However, in the presence of glucose insulin has the reverse effect, i.e., enhancing chemo-attraction to glucose. This effect of insulin on glucose chemotaxis indicates that the regulatory circuitry for glucose metabolism and chemotaxis may be more complex than previously thought [32] .
Based on the observations made in this study, human insulin appears to differentially regulate expression of virulence factors dependent on the presence of glucose. In the absence of glucose, insulin appears to act in a warning mode as a chemo-repellent seemingly signaling an inhospitable environment for colonization and promoting a planktonic population. In contrast, with glucose present insulin appears to signal a welcoming environment for colonization and long term survival by enhancing expression of adherence properties. This control of environmental adaptation would be crucial for environmental survival perhaps functioning through modulation of the CsrA/CsrB and BarA/UvrY systems which are part of the glucosemediated regulatory circuitry for virulence factor expression, including biofilm formation [33] .
The possible role this insulin-mediated switching of virulence factor expression plays in asymptomatic bacteriuria vs. the higher rate of urinary tract infections occurring in patients with type 2 diabetes and in females with gestational diabetes as compared to type 1 diabetics remains to be determined.