GENOTYPIC CHARACTERIZATION OF TOXINS AND IRON ACQUISITION AGENT AS VIRULENCE FACTOR OF UROPATHOGENIC ESCHERICHIA COLI.

SM.Nachammai, KarthikaJayakumar, Vinithra Suresh and Kousalya.M. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 11 September 2018 Final Accepted: 13 October 2018 Published: November 2018

Escherichia coli called as UPEC possessing specific urovirulence factors, helps in adhesion, survival of the bacteria and leading to the infection in the urinary tract by surpassing the host immune response. Certain virulence factors are responsible for survival of the bacteria which is needed for long term persistence of the bacteria to cause recurrent infections. Aerobactin toxin helps in scavenging the iron from the host, while hemolysin A and cytotoxic necrotizing factor suppresses the inflammatory process and causes tissue damage. The aim of this study is to determine the presence of these fitness and virulence factor genes in the E. coli isolated from urine. Invasive toxins like α-hemolysin (hlyA), cytotoxic necrotizing factor (cnf) and an iron chelating agent -aerobactin (aer) are certain characteristics of UPEC (1) . hlyA and cnf operons encodes for exotoxins which are membrane active and stimulates PMN's cytoxicity respectively causing tissue damage and dysfunction of local immune responses. CNF toxin are mostly coproduced with alpha hemolysin, enhances the adherence to urothelial cells. The battle wages will always be present between the pathogens and iron limited host. Free iron (Ferric, Fe 3+ cations is essential for the survival of pathogenic organism in its host. This iron-limited host cell environment is favoured to UPEC by an iron chelator -aerobactin which scavenges the ferric ions from the parasitized host (2) .

Alpha hemolysin (HlyA) and its action in UTI:
It's a 107 kDa a membrane active protein induces hemolysis by creating 2nm wide pores in the erythrocyte membrane by increasing the permeability and swelling of cells leading to ruputure of RBCs which can be demonstrated invitro using Blood agar showing beta hemolysis. It's a Gly-rich nanopeptide protein repeated in tandem between 9 and 42 times which is named as RTX (Repeat in ToXin) (3) .
hlyA binds to the hydrophobic surface of the bilayer membrane at neutral pH and lytic action on cell membrane requires calcium ion. E.coli uses endogeneous amplification, PX channel receptor especially PX 1, PX7 activation induces hemolysis in addition to that pannexin1, a pore forming protein in macrophages is a recently identified protein which increases the permeability for hemolysis (4) .

Cytotoxic necrotizing factor type 1 (CNF1) and its action in UTI:
110-kDa protein toxin induces actin reorganization into stress fibers and retraction fibers in human epithelial cells allowing them to spread, mediates cellular effects by the activation of small GTP-binding proteins. It stimulates PMNL cytotoxicity and enhances the effect on adherence to uroepithelial cells as well as the production of radical oxygen products. CNF-1 decreases the phagocytosis of PMNL cells and facilitates the growth of bacteria (5) .

Aerobactin as an iron acquisition agent:
Iron acquisition is a critical requirement for UPEC survival in an environment that is iron-limited as the urinary tract. Aerobactin is one of the siderophore, a small-molecule iron chelators that are produced by UPEC strains to scavenge ferric iron (Fe 3+ ), an insoluble free cation, as free iron concentration are low in mammalian hosts. . Bacteria retrieve iron-bound siderophores through receptors that facilitate the transport of siderophore-iron complexes through the bacterial membrane and into the cytosol where the iron is released. Limiting iron availability is an important host defense against invading bacterial pathogens. These are some of the urovirulence characteristics that aid in colonization, host evasion and survival of UPEC strains (6) .

Materials and Methods:-Bacterial isolates:
The total of 212 E. coli isolated from urine samples were confirmed by biochemical tests based on standard bacterial identification.

DNA extraction:
DNA extraction was performed using boiling lysis method. Before DNA extraction, the E. coli isolates were cultured in LB broth at 37 o C for 18 hours. Bacteria were pelleted from 1.5 ml LB broth then suspended in 200 ml of sterile deionized water and incubated at 100 o C for 10 min. After centrifuging, the supernatant was used as template DNA and stored at -20 o C PCR amplification: PCR amplification of virulence genes were used to reveal the prevalence of three virulence genes hlyA, cnf, aer using specific primers. The amplification of virulence genes was carried out in a Thermal Cycler (Eppendorf Master) after standardizing the PCR conditions, consisted of initial denaturation at 94 o C for 2 min, followed by 30 cycles of denaturation at 94 o C for 1 min annealing at 60 o C for 30s and extension at 1 min 30s and final extension at 72 o C for 5min (8) . PCR product were then loaded in 1% agar gel electrophoresis and amplified DNA fragments were detected by UV fluorescence transilluminator and the size of the amplicons was estimated by comparing with 1kb DNA ladder included in the same gel.

Results:-
The results of molecular detection of specific toxin genes in E. coli were, aer (iron acquisition factor) accounting up to 107(50.5%), 74 (34.9%) and 33 (15.6%) isolates possess cnf and hlyA genes respectively. None of these were present in 73 (34.4%) of the isolates. The single isolate possess one gene or association of two or more genes, the expression of it optimally or sub-optimally makes the long persistence in the host and increase in severity of the infection.    HlyA (Alpha hemolysin), a pore forming toxin confers stable insertion into the epithelial and macrophage membranes which degrades the cyto-skeletal scaffolding paxillin protein and regulatory proteins of natural killer cell signaling pathway. HlyA induces the proteolysis of host proteins as well as modulates the function of epithelial cells, disables macrophages and finally inflammatory responses get suppressed (4) .
CNF1 and HlyA are commonly found in UPEC isolates and are co-expressed as closely linked genes. CNF1 is a 115-kDa exotoxin has the role in pathogenesis of neonatal meningitis. Studies states that CNF has expressed in 31-44% of cystitis and 36-48% of pyelonephritis, it activates Rho GTPases, contributing to urothelial cell invasion by inducing the actin stress fiber formation, lamellopodia, filopodia and the inflammatory signal pathway gets modulated resutkting in cytotoxic effect (5) .
aer (Aerobactin), a hydroxamate siderophore, produced maximally at low pH, has a higher Fe 3+ binding stability in acidic environments (11) . Iron is a known compound to increase the growth of the bacteria, highly expressed fitness factor commonly expressed in all UTI patients and negatively regulated by ferrous iron for the uptake of ferric in low iron conditions (12) . It is an important factor in the progression of pyelonephritis (13) .

Conclusion:-
These virulence factors play an essential role in uropathogenecity; also the frequency of its expression increases the severity of the disease with recurrent infection and may also be responsible for kidney damage and other extra intestinal manifestations.