Objective
The mechanism of infection-related deaths of pregnant rats and intrauterine growth restriction are not understood. We assessed whether nitric oxide (NO) has differential effects on infection with Escherichia coli Dr/Afa mutants that lack either AfaE or AfaD invasins.
Study Design
Sprague-Dawley rats were infected intrauterinally with the clinical strain of E coli AfaE + D + or 1 of its isogenic mutants in the presence or absence of the NO synthesis inhibitor N G -nitro-L-arginine methyl ester (L-NAME). Maternal/fetal mortality rates, fetoplacental weight, and infection rates were evaluated.
Results
Maternal and/or fetal death was associated with the presence of at least 1 virulence factor (AfaE + D + >AfaE + D − >AfaE − D + ) and was increased by L-NAME treatment. The fetal and placental weights were lower than controls and were further reduced by L-NAME treatment.
Conclusion
These results demonstrate that NO enhanced AfaE- and AfaD-mediated virulence and plays an important role in Dr/Afa + E coli gestational tropism.
Urinary tract infection is a major cause of fetomaternal morbidity during pregnancy. Escherichia coli is the major etiologic agent of gestational urinary tract infection (asymptomatic bacteruiria, cystitis, and pyelonephritis), which accounts for 90% of infections. Different clinical forms of urinary tract infection are associated with multiple complications. Asymptomatic bacteruiria is associated with low birthweight; therefore, all pregnant patients require prenatal screening and antibiotic treatment. Urinary tract infection, which includes pyelonephritis, has been implicated in preterm labor, cerebral palsy, septicemia, and maternal death. We proposed that urinary tract infection complications develop in part because of gestational tropism of E coli. Our laboratories pioneered studies on gestational tropism of E coli –producing adhesins of the Dr family (Dr/Afa + E coli ) and pyelonephritis. We established an E coli experimental uterine infection model in pregnant rats and showed that Dr adhesin is a key lethal factor in pregnant, but not nonpregnant, rats. However, the impact of Dra E and Dra D, which together form Dr adhesion, on lethality and fetal growth and the role of nitric oxide (NO) in the host defense are not well understood.
The strains of E coli that predominantly are associated with gestational urogenital infection are those of the O75 serotype that frequently produced adhesins of the Dr family, which play an essential and critical role in the infectious process and gestational virulence. Dr/Afa + E coli account for 40% of pyelonephritis cases in the third trimester of pregnancy. The adhesins are encoded by very similar operons of at least 5 genes and are associated with fimbriae (such as Dr, Dr-II, and F1845 adhesins) and small fibrils (such as AfaE-I and AfaE-III adhesins). These adhesive structures are heterologous polymers that are composed of a Dra/AfaE fiber and a Dra/AfaD tip protein. We previously reported that decay-accelerating factor (DAF), which also is called CD55, serves as a tissue receptor for Dr/Afa + adhesins, that the β1 integrin is recognized by the Dra/AfaD proteins, and that both E and D may contribute to invasion.
NO, which is a free gas molecule, is reported to be involved in a variety of physiologic processes that include neurotransmission, vasodilation, smooth muscle relaxation, and infection. NO is generated constitutively from L-arginine by NO synthase (NOS); 3 different isoforms of NOS (NOS I, II, and III) have been identified in mouse, rat, and human. Although the role of NO in the host defense is not well understood, reports that have used experimental animal models or clinical patients strongly suggest an antibacterial activity of NO. Inhibition of NO aggravates Staphylococcus aureus arthritis in mice, reduces survival time after an E coli challenge in rats, and demonstrates a detrimental effect in the murine peritonitis model. It has also been shown that the NO donor compounds do not affect the viability and survival of bacteria themselves but enhance the neutrophil’s ability to kill E coli , Proteus vulgaris , and Salmonella anatum . The findings from our laboratories further showed an inverse relationship between the severity of experimental pyelonephritis and NO production in C3H/HeJ mice. In addition, in clinical patients with infectious diseases, NO production appeared to be increased in macrophages and was correlated with better clinical outcome. NO production and 3 NOS isoforms were reported to be present in rat uterine tissues, and elevated NOS II expression was shown to contribute to the increased NO production and consequent uterine quiescence during pregnancy. In our previous studies, we reported that pregnancy modifies NO-associated host resistance mechanisms and alters the risk for genitourinary infection by Dr/Afa + E coli and its complications. Inhibition of NO synthesis during pregnancy doubled the mortality rate of the Dr/Afa + E coli infected rats, which indicates that endogenous NO may play a role in the host’s ability to eliminate bacteria and lessen sepsis.
In this study, we investigated both microbial and host factors that are involved in the mechanism of gestational tropism. We hypothesized that both Dr/Afa + E coli and NO contribute to the gestational tropism. It has been suggested that the invasive capacity of Dr/Afa + E coli depends on 2 virulence factors (Dra/AfaE and Dra/AfaD proteins) that are responsible for adhesion and internalization that are mediated by 2 different receptors. However, the contribution of either Dra/AfaE or Dra/AfaD subunit to in vivo infection is poorly understood. In our previous studies, we showed that experimental intrauterine infection with Dr/Afa + E coli is lethal to pregnant, but not to nonpregnant, rats. We sought to assess the influence of genes that encode the 2 proteins not only on maternal mortality rates but also on fetal and placental growth rates. All previous studies have analyzed NO-associated changes in infection that are caused by E coli that bears intact Dr/Afa structure, which was assumed to be essential for the virulence of E coli . We investigated whether the NO inhibitor N G -nitro-L-arginine methyl ester (L-NAME) has differential effects on the maternal mortality rates and fetoplacental growth rates in rats with experimental intrauterine infection with the use of AfaE and AfaD mutants of E coli .
Materials and Methods
Bacterial strains
The strains used in this study are listed in Table 1 . E coli A30 is a clinical isolate that expresses the afa-3 operon. A30 derivatives that contain mutated afa E3, afa D3, or both afa E3 and afa D3 genes were constructed by allelic exchange as previously described. The growth rates were similar for all these strains.
| Bacterial strain | Relevant characteristics | Mannose-resistant hemagglutination | Reference |
|---|---|---|---|
| A30 (AfaE + D + ) | Cystitis-associated isolate carrying the afa -3 operon | + | 49 |
| AL858 (AfaE − D + ) | Isogenic mutant of A30 with a mutated afa E3 gene | − | 48 |
| AL861 (AfaE + D − ) | Isogenic mutant of A30 with a mutated afa D3 gene | + | 47 |
| AL863 (AfaE − D − ) | Mutant of AL858 with a mutated afa D3 gene | − | 47 |
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
