Enterobacteriaceae

Inner Membrane

The inner—or cystoplasmic—membrane is a phospholipid bilayer that maintains the proton motive force for energy storage and regulates the passage of molecules to and from the cytoplasm. The inner membrane may feature hundreds of proteins, including integral membrane proteins with transmembrane domains traversing the bilayer, lipoproteins inserted in the outer leaflet, and peripheral membrane proteins in either the inner or outer leaflet. The inner membrane includes the proteins involved in electron transfer and oxidative phosphorylation, and the F ₁ F ₀ adenosine triphosphatase that couples proton transport to adenosine triphosphate (ATP) synthesis. Furthermore, the inner membrane may feature efflux pumps, solute transporters, protein translocation systems, polysaccharide export systems, and histidine kinase signaling proteins.

Periplasmic Space

The periplasmic space between the inner and outer membrane is an aqueous, oxidizing environment containing the peptidoglycan cell wall and a high concentration of proteins. These proteins include oxidoreductases, isomerases, chaperones, proteases involved in protein folding and degradation, lipoprotein-sorting proteins, detoxifying enzymes, and solute-binding proteins that ferry molecules across the periplasmic space. The periplasmic space also features enzymes involved in the biogenesis of peptidoglycan, lipopolysaccharide (LPS) and the capsule.

Peptidoglycan Cell Wall

The gram-negative cell wall is a thin layer of peptidoglycan—also called murein—composed of alternating N -acetylglucosamine and N -acetylmuramic acid amino sugars joined by β-1,4 linkages with a short peptide composed of l -alanine, d -glutamic acid, l - meso -diaminopalmelic acid, and d -alanine attached to the carboxyl group of the muramic acid. Linear strands of peptidoglycan are linked by amide bonds to adjacent strands, with murein lipoprotein anchoring the layer to the inner leaflet of the outer membrane. The peptidoglycan layer of each bacterium is believed to comprise a single contiguous molecule enveloping the organism, responsible for shape and osmotic stability yet undergoing constant remodeling as the organism elongates and divides.

Outer Membrane

The outer membrane is an asymmetrical lipid bilayer featuring a mostly phospholipid inner leaflet and an outer leaflet composed primarily of LPS. The outer leaflet also features porins—membrane proteins with a conserved β-barrel fold enclosing a central aqueous channel—that regulate the passage of hydrophilic molecules. The polarity of LPS in the outer leaflet prevents penetration of lipophilic molecules, including detergents, dyes, and hydrophobic antimicrobials.

Because LPS is an essential component of the outer membrane of all gram-negative bacteria, some argue it is not a virulence factor per se. Regardless, LPS will be discussed in more detail later; the repeating oligosaccharide attached to the LPS core is referred to as the O antigen, which is the basis for serogroup classification. Each species within the Enterobacteriaceae has multiple O-antigen types.

Other Surface Polysaccharides and Capsules

Other potential surface polysaccharides of the Enterobacteriaceae family include enterobacterial common antigen, colonic acid, and a wide variety of polysaccharide envelopes known as capsules. Enterobacterial common antigen is remarkably conserved throughout the family, suggesting an important although poorly understood function. Many of the Enterobacteriaceae—including E. coli strains—feature a colonic acid layer, which resembles a subset of capsule types. True capsules are linked to either LPS or α-glycerol phosphate and are the basis of the K-antigen serotyping scheme. Some enterobacterial capsules can be quite luxuriant, imparting the highly mucoid colonial morphology to Klebsiella and Enterobacter species, for example.

Flagella

Flagella are flexible, rotating surface appendages that propel bacteria through liquid environments. Most members of the Enterobacteriaceae are motile by way of flagella, often emanating from all sides of the organism ( Fig. 218.2A ). Even nonmotile Enterobacteriaceae sometime retain genes specific to flagellar expression and are then capable of motility under certain conditions. Like LPS, flagellin is recognized by host innate immune system pattern recognition receptors, which can lead to neutrophil recruitment and initiation of an inflammatory response.

Flagellar biogenesis proceeds from base to tip and is orchestrated by a complex regulatory network, responding to a variety of extracellular signals. The flagellar filament is composed of a hollow helical array of the protein flagellin whose amino and carboxyl termini is highly conserved within and across a species. In contrast, the surface-exposed middle of the flagellin molecule is highly variable as a result of both diversifying mutations and recombination after horizontal gene transfer. This diversity is represented in the H-antigen typing scheme, the third component of O:K:H serotyping.

Pili

Most of the Enterobacteriaceae produce thin nanofilaments extending from the bacterial surface called pili—also known as fimbriae—that mediate autoaggregation and adhesion to host cells. Furthermore, pili facilitate bacterial conjugation and are often encoded by plasmids—capable of harboring virulence and antimicrobial resistance genes—to mediate intercellular contact and genetic exchange. The family Enterobacteriaceae features a variety of pilus types, which differ in morphology and function. An individual strain may produce multiple different pili even of the same type.

Chaperone-usher type pili are common among the Enterobacteriaceae and include the ubiquitous type 1 pili (see Fig. 218.2B ). Assembly begins with secretion machinery exporting subunits of the major structural protein pilin to the periplasmic space to bind with chaperones to prevent premature subunit interactions. The pilin-chaperone complexes are then delivered across the outer membrane—in a specific order starting with the tip—by a membrane channel protein known as the usher. Most chaperone-usher type pili feature a rigid rod composed of a helical array of pilin joined end to end with a thinner, more flexible tip. The tip often features adhesin proteins that serve as critical virulence factors.

Type IV pili are also widespread among the Enterobacteriaceae, often forming ropelike bundles expressed at the poles of the organism (see Fig. 218.2C ). The type IV pilin protein is processed to its mature form by a dedicated prepilin peptidase that also N -methylates the amino-terminal residue. Type IV pili are retractable, which facilitates aggregation and disaggregation and a type of locomotion called “twitching motility.”

Adhesins

Many adhesins of the Enterobacteriaceae—including pili, outer membrane proteins, and even surface carbohydrates—have proven indispensable to infection. An organism may feature a number of adhesins at once or in sequence in response to environmental cues or as a result of random phase variation.

Type 1 pili are the prototypical enterobacterial adhesin, composed of a rigid rod of repeating subunits of the protein FimA arranged in a helical array. The pilus tip contains a short fiber composed of FimG and FimH subunits—the so-called fimbrial adhesins—joined end-to-end with the FimA rod. FimH binds to mannose residues found in glycoproteins and glycolipids on the surface of host cells. Type 1 pili expression is subject to phase variation because of the invertible DNA element flanking the promoter of the fim operon, resulting in variable pili production depending on the organism's environment. Furthermore, type 1 pili adhesion is enhanced by shear force such as the flow conditions of the genitourinary tract. In addition to being well-established as virulence factors, type 1 pili are also important in colonization and transmission. Furthermore, a given enterobacterial strain may produce multiple other chaperone-usher type pili and type IV pili, discussed in more detail later, depending on the particular species.

The invasin-intimin family of proteins are the best characterized outer membrane adhesins, described from studies of Yersinia pseudotuberculosis and enteropathogenic E. coli (EPEC). These proteins—inserted into the bacterial outer membrane—share common structural features with similar amino-acid sequences, especially in their amino-terminal and central domains. The membrane portion is connected by a flexible hinge region to a rigid rod of repeating subunits similar in structure to portions of an immunoglobulin molecule. The carboxyl-terminal adhesin domain shares similarities to calcium-binding lectin molecules.