During UTIs, UPEC pathogenesis includes the following: (1) colonization of the periurethral, urethral, and vaginal areas; (2) ascent into the bladder lumen and growth as planktonic cells in urine; (3) adherence to the surface and interaction with the bladder epithelium defense system; (4) biofilm formation; (5) invasion and replication by forming bladder intracellular bacterial communities (IBCs), where quiescent intracellular reservoirs form and reside in the underlying urothelium; and (6) kidney colonization and host tissue damage with an increased risk of bacteremia/septicemia [12].

UPEC colonize the bladder using diverse virulence factors that play critical roles in the pathogenesis of UTI: surface structural components, such as lipopolysaccharide (LPS), polysaccharide capsule, flagella, pili, non-pilus adhesins, curli, outer-membrane vesicles, outer-membrane proteins, secreted toxins, secretion systems, and TonB-dependent iron-uptake receptors, including siderophore receptors [13].

LPS are amphipathic molecules, consisting of fatty acids lined to an oligosaccharide core, stuck to a long polysaccharide chain, commonly called the O antigen. LPS mediate multiple aspects of the UPEC life cycle, including the ability to acutely colonize bladders, fashion reservoirs, and arouse the innate and adaptive immune responses [14]. Moreover, LPS provides resistance against hydrophobic antibiotics, such as some antibiotics and bile salts, which form when LPS is decreased at the cell surface [15].

In UPEC, the fim operon encodes type 1 pili exhibiting mannose-sensitive hemagglutination, whereas the pap operon encodes P- or Pap-pili that are capable of interacting with the digalactoside unit in the P-blood group antigen. The chaperone-usher pathway (CUP) assembles the pili. Type 1 pili are tipped with the adhesin FimH, which recognizes mannosylated uroplakins and α1β3 integrins with stereochemical specificity, initiating colonization and invasion into umbrella cells [16]. Type 1 pili, combining these cells, triggers a signal transduction cascade, activating Rho GTPases, such as those from the Rac family, causing actin rearrangement, and internalization of UPEC via a zippering mechanism composing a plasma membrane sheathe that engulfs the bacterium [17]. Moreover, the expression of type 1 pili is administered stringently by a phase variation, which reversibly diverts between the type 1 pili active expression (Phase-ON, piliated cells) and a loss of expression (Phase-OFF, non-piliated cells). Environmental signs within the urinary tract, such as salt growth conditions and acidic pH, manage stringently the molecular pathways involved in reversible diverting between the ON-OFF Phases [18].

Unlike the mannose-binding adhesion (FimH) of type 1 pili, the adhesin of P pili (PapG) sticks globosides containing glycolipids that are present in the kidneys [19]. In addition, PapG modulates the local secretory-antibody immune response by interacting with TLR4 to reduce the level of polymeric immunoglobulin receptor expression, impairing immunoglobulin (Ig)A transport through the lamina propria and epithelial cells to the kidney lumen. This mechanism helps UPEC evade a key host protective mechanism, allowing the establishment of ascending infection [20].

Curli are bacterial surface appendages that secrete subunits from the cell as soluble monomeric proteins and possess the typical structure and physical characteristics of amyloid fibrils. In UPEC, proteins encoded in the csgDEFG operon organize curli formation. The operon accessory proteins, CsgE, CsgF, and CsgG, are required to promote the secretion of CsgA, whereas CsgB nucleates CsgA subunits into curli fibers [21].

While pili are involved in the first attachment of UPEC to the urinary tract mucosa, UPEC employs several other afimbrial adhesins. The adhesin TosA was found in approximately 30% of urinary tract isolates [22]. Another adhesin FdeC aided colonization of the kidneys and bladder in a mouse infection model [23], whereas the iron-regulated adhesin (Iha) mediates adherence to bladder epithelial cells [24].

UPEC fabricates three main types of toxin: α-hemolysin (HlyA), cytotoxic necrotizing factor 1 (CNF1), and secreted autotransporter toxins [9]. HlyA forms pores in the umbrella and promotes their lysis, facilitating iron and nutrient acquisition by the bacteria. HlyA also triggers exfoliation, exposing deeper layers of the uroepithelium for colonization and promoting bacterial spread to other hosts following cell expulsion in the urine [2526]. CNF1 influences actin remodeling in the host cell through three small RHO GTPases: RAC1, RHOA, and cell division control. CNF1 enters the host cell in endocytic vesicles, by sticking to the basal cell adhesion molecule receptor, constitutively initiating RHO GTPases via the deamination of a glutamine residue; this generates actin cytoskeletal rearrangements and membrane ruffling, guiding increased bacterial internalization [27]. CNF1 is not necessary for infection but might be a fitness factor [9]. UPEC also secretes autotransporter toxins. Sat (secreted autotransporter toxin) is a serine protease that intercedes in the cytopathic effects on the kidney and bladder cell lines in vitro and causes tissue damage and immune responses in infected mice [28]. The autotransporter, Pic, with serine protease activity, is expressed during infection, but it does not aid in colonization. Another autotransporter Tsh, which lacks detectable serine protease activity, is expressed during infection in mice [29].

Similar to adherence, motility is another trait often associated with bacterial virulence. Complex surface structures called flagella mediate motility. UPEC is capable of flagella-mediated motility, which contributes to the fitness of UPEC during a UTI and the ascension of infection [30].

Iron, an essential nutrient, is isolated by the host, and successful bacterial pathogens must have the means to acquire it. Therefore, iron acquisition is a crucial requirement for UPEC survival. Consequently, IBC UPEC often shows the upregulation of redundant systems for iron acquisition. Siderophores are small molecule iron chelators provided by UPEC strains to scavenge ferric ions (Fe³⁺). UPEC produces several siderophores, of which aerobactin and yersiniabactin are essential in the urinary tract [1331]. Aerobactin is expressed strongly, stable at low pH, and displays higher iron sticking capacity than enterobactin. Yersiniabactin is important in bio-film formation in urine and protects against intracellular killing by copper stress because it sequesters host-derived copper [32].

Pathogens can usually scavenge Fe from precursors, such as heme and hemoglobin. UPEC contains two outer membrane heme receptors, ChuA and Hma. Heme uptake via these receptors contributes to fitness during a UTI; the hma mutant outcompetes the chuA mutant, indicating that ChuA is the predominant heme transporter [33]. Moreover, ChuA aids in the intracellular growth of UPEC in bladder epithelial cells. During intracellular growth, chuA is expressed strongly, and its mutant fails to grow at the wild-type level. Heme uptake systems contribute to fitness during extracellular and intracellular growth in UPEC [34].

Similarly, zinc is another essential nutrient sequestered by the effectors of nutritional immunity. Two distinct zinc import systems, ZnuACB and ZupT, operate in UPEC. Indeed, the high-affinity zinc-transport system, ZnuACB, contributed to the fitness of UPEC during experimental UTI [3536].

Another important factor that supports host colonization is the ability to evade the host immune response. Iron is a crucial nutrient that must be obtained by UPEC during an infection, and enterobactin represents one mechanism of iron procurement. On the other hand, the host protein lipocalin-2 issued by neutrophils specifically sticks to, and sequesters enterobactin, making it unable to provide UPEC with iron. Interestingly, UPEC can revise enterobactin by glycosylation; glycosylated enterobactin, known as salmochelin, are not identified and sequestered by lipocalin-2, enabling UPEC to escape the host defense mechanism mediated by lipocalin-2 [937].

In addition, some UPEC strains encode the proteins, SisA and SisB, which suppress the host inflammatory response during the early stages of infection. A deletion of functional copies of these genes in UPEC causes significantly more inflammation in the host. Furthermore, a phase variation of type 1 fimbrial genes might assist UPEC in evading the immune system [38].

After the first attachment, P. mirabilis produces mannose-resistant Proteus-like (MR/P) pili (CUP pili) that activate biofilm formation and colonization of the kidney and bladder; these are critical for catheter-associated biofilm formation. The most understood are possibly MR/P pili, which are synthesized in vivo, eliciting a strong immune response during infection; these have been implicated in auto aggregation and biofilm formation, contributing to virulence [39]. Other CUP pili encoded by P. mirabilis include P. mirabilis-like fimbriae (PMF), which are important for bladder and kidney colonization, non-agglutinating fimbriae (NAF) for attachment to uroepithelial cells in vitro, and P. mirabilis P-like fimbriae, which are recognized from a canine UTI isolate. Interestingly, the genes encoding this fimbria have also been found in human clinical isolates. On the other hand, the in vivo mechanistic roles of PMFs, NAFs, and their receptors remain unclear [4041].

P. mirabilis fabricates two toxins: hemolysin (HpmA) and Proteus toxic agglutinin (Pta), which have been implicated in tissue damage and dissemination to the kidneys, initiating acute pyelonephritis [42]. Boht HpmA and Pta induce tissue damage during infection. On the other hand, Pta alone promotes the ability of P. mirabilis to colonize the urinary tract [43].

HpmA is a Ca⁺-dependent pore-forming cytolysin that destabilizes the host cell by inserting itself into the cell membrane, causing Na⁺ efflux [5]. On the other hand, the surface-associated cytotoxic protease, Pta, is functional only in alkaline pH, such as that generated by the activity of P. mirabilis urease. In the proposed mode of action, Pta penetrates the host cell membrane, causing osmotic stress, the leakage of cytosol, and the depolymerization of actin filaments. Therefore, the structural integrity of the cell is compromised, resulting in kidney and bladder damage. Moreover, Pta induces bacterial cell–cell interactions via autoaggregation [54144].

Perhaps the most defining hallmark of P. mirabilis is swarming motility, a specialized form of flagella-mediated movement that requires the differentiation of cells into an elongated morphotype teeming with flagella. These elongated cells appear to be limited during infection, but P. mirabilis is capable of swarming across the surface of urinary catheters. Swarming and swimming motility during infection are difficult to distinguish because both are flagella mediated. Strains lacking flagella were weakened in a murine model, indicating a role for flagella during ascending UTI. On the other hand, a strain of P. mirabilis lacking flagella has been separated from a human patient, suggesting that flagella are not mandatory for its virulence [945].

Historically, it was believed that P. mirabilis did not produce siderophores, unlike other members of the Enterobacteriaceae family. On the other hand, a microarray-based transcriptional study of P. mirabilis, cultured under Felimited conditions, revealed genes involved in Fe uptake, including siderophore systems, heme receptors, and receptors for exogenous siderophores. This study also led to the identification of proteobactin, a novel siderophore system, and a yersiniabactin-related siderophore system in P. mirabilis [46], which were developed for Fe³⁺ acquisition. The yersiniabactin-related siderophore was originally recognized in a signature-tagged mutagenesis screen designed to detect the virulence factors in a murine UTI model and found to induce the successful colonization of the urinary bladder [34]. Moreover, P. mirabilis utilizes HmuR1 and HmuR2, which are outer membrane receptors that activate heme import. A loss of HmuR2 attenuated this process in both the kidney and bladder in a murine UTI model [47].

Similar to UPEC, the ZnuACB high-affinity zinc transport system promotes Zn uptake in P. mirabilis, both in vitro and in vivo. A znuC mutant with a Zn²⁺ limitation was damaged during growth and contained a fitness defect during a UTI in a mouse model. Overall, Zn²⁺ uptake by the ZnuACB system is involved in urofitness in both UPEC and P. mirabilis [48].

At least three mechanisms are used by P. mirabilis for immune evasion during a UTI. The first is the production of ZapA, a zinc metalloprotease capable of cleaving several host proteins, including serum and secretory IgA1, IgA2, and IgG, antimicrobial peptides (AMPs), and complement proteins [49]. ZapA activity was detected in the urine of infected patients, probably helping P. mirabilis colonize the urinary tract [949]. In the second mechanism, P. mirabilis possibly utilizes MR/P fimbriae, which are capable of undergoing phase variation, to facilitate immune response avoidance. In the third mechanism, flaA is the predominate version of flagellin expressed by P. mirabilis, whereas flaB is normally silent. On the other hand, upon recombination of these genes, P. mirabilis produces antigenically distinct flagella, enabling it to subvert the immune response [59].

UTI caused by P. mirabilis is characterized by urolithiasis and is affected by urease, a Ni²⁺-dependent metalloenzyme crucial for colonization of the kidney and bladder [3941]. The P. mirabilis urease is expressed constitutively during growth in urine. The urease actively hydrolyzes urea into ammonia and carbonate several times faster compared to those by other species, such as Proteus vulgaris, Providencia rettgeri, and M. morganii. The highly active P. mirabilis urease produces rapid crystal formation, which is trapped within the polysaccharides produced by the attached bacterial cells. They form crystalline biofilms on catheters [50], affording protection to P. mirabilis from the host immune system and antibiotics. Moreover, these structures block urine drainage from the ureters, potentially resulting in reflux and activating the progression to pyelonephritis, septicemia, and shock [41].