Bacterial Infection of the Host

The infection process:

1.        Exposure to pathogens

2.        Adherence to skin/mucosa

3.        Invasion through epithelium

4.        Multiplication – growth and production of virulence factors and toxins

The disease process:

1.        Toxicity – toxin effects are local/systemic

OR

1.        Invasiveness – further growth of original and distant sites

2.        Tissue or systemic damage

Exposure – entry points:

-            Respiratory tract

-            Gastrointestinal tract

-            Urogenital tract

-            Breaks in the skin surface

Mucous membranes line the digestive, respiratory, and reproductive tracts. Ita a primary barrier protecting the body from the external world. It’s a key site of innate immune defence. It has no particular specificity for any one pathogen. There are several immune functions at the mucous membranes:

-            Lysozymes hydrolyse the bonds in the sugar backbone of the peptidoglycan

-            Mucociliary clearance helps expel inhales microbes from airways

-            Cationic antimicrobial peptides disrupt bacterial membranes

-            Mucosa-associated lymphoid tissue (MALT) provides accumulation of lymphocytes

Adherence:

Numerous bacterial components contribute to adherence, including:

-            Pili and fimbriae. They are short, hair-like projections that cover the cell. They are the bacterial cell structure that bind host cell surface glycoproteins. Pili are typically longer and fewer in number than fimbriae, and some pili also function in the process of conjugation.

-            Capsules. They are most commonly comprised of polysaccharides, located outside of the cell envelope. It can be found in both Gram positive and negative organisms. They promote attachment through specific molecular interactions, as well as being inherently ‘sticky’. They also contribute to immune evasion.

-            Lipoproteins. Membrane proteins that are anchored to membranes through an N-terminal lipid moiety attached to a conserved Cysteine.

Bacterial cells will possess multiple ‘adhesins’ that bind different host cell ligands.

Salmonella enterica, serovar Typhi (Salmonella typhi):

It’s a causative agent of typhoid fever. Type IV pili of S. typhi enable the attachment to epithelial cells, and subsequent entry. The pili bind to host CFTR protein (Cystic Fibrosis Transmembrane conductance Regulator). Numerous population genetic studies have confirmed a correlation between cftr polymorphisms and partial protection from typhoid fever

Adherence and biofilms:

Biofilms are an aggregate of bacterial cells attached to a surface and encased in a matrix. Matrix components are produced by the bacteria themselves. During infection, host components can also contribute to biofilm matrix.

Biofilm significance:

Clinical significance – they offer protection against immune system and antibiotics, chronic infection. Bacteria in biofilms can be up to 1,000 times more tolerant to antibacterials than the equivalent planktonic bacteria and contribute to failure of antimicrobial therapy. Tolerance is a transient non-heritable phenotype. Tolerance mechanisms include:

-            Restricted diffusion of antimicrobials through the biofilm matrix

-            Reduced metabolic activity and growth rates

-            Presence of persister cells – effectively dormant cells. Antibiotics target metabolically active bacterial cells – no activity = can’t work/target cells. Metabolically active cells are directed to the periphery of cells.

Many antimicrobial agents can readily diffuse through the biofilm matrix, however slowed diffusion may enable microbes to mount an adaptive response. Diffusion is profoundly influenced by charge interactions with matrix components. Positively-charged aminoglycoside antibiotics can bind matrix components, while negatively-charged B-lactam antibiotics do not.

Industrial significance – biofilm growth within industrial plants and pipelines can impact processes and present contamination risks.

Adherence of bacteria to host cells can also lead to the invasion of host cells, which is often mediated by cytoskeletal rearrangements.

Invasion:

The consequences of invasion can be wide-ranging, and dependent on the specific nature of the host-pathogen interaction, It triggers:

-            The production of proinflammatory cytokines and chemokines, leading to the recruitment of inflammatory cells

-            Epithelial cell apoptosis and subsequent exfoliation – helps clear infection, clearance mechanism

-            Persistence of the bacterium within the host cell

-            Invasion of underlying cells/tissue – e.g. Salmonella typhi

Flagellar-mediated motility:

Flagella are long, thin appendaged attached to the bacterial cell. They function by rotation, pushing or pulling the bacterial cell through the liquid medium. The speed of flagellar rotation varies depending on the strength of the proton motive force. Certain bacteria can sense environmental signals and move away or towards them by altering the direction of flagella rotation.

Chemotaxis, and the ‘biased random walk’:

-            Counter-clockwise flagellar rotation results in the cell swimming forward

-            Clockwise rotation results in the cell stopping and randomly ‘tumbling’

If moving up a gradient of attractant, the runs become longer and tumbles less frequent -> ‘biased random walk’.

Iron is an essential co-factor for numerous basic metabolic pathways in both the mammalian host and microorganism. It is a critical component of cytochromes and iron-sulphur proteins of the ETC, and an essential co-factor for many enzymes. The competition for iron between pathogens and host is of critical importance for pathogenesis.

Iron can exist in two oxidation states – ferrous (Fe²⁺), and ferric (Fe³⁺). Ferric dominates in oxygenated environments and at a neutral pH, whereas ferrous dominates in anaerobic environments and at low pH. The solubility of Fe3+ is extremely low compared to Fe2+. Bacteria must either:

1.        Reduce Fe3+ to the more soluble Fe2+

2.        Employ ferric iron chelators as solubilising agents

A first line of defence against infection is the withholding of nutrients to prevent pathogen growth.

The majority of iron in the mammalian host is intracellular:

-            Sequestered by the iron storage protein, ferritin

-            Complexed with haem as a cofactor of haemoglobin or myoglobin

Availability of extracellular iron is minimal:

-            Extracellular environments are typically aerobic and neutral pH

Under aerobic conditions, iron can be extremely toxic through its interaction with reactive oxygen species, meaning all aspects of iron homeostasis are tightly coordinated. Keeping iron bound reduces its toxicity. The toxicity comes from 2 main reactions – iron reduction, and Fenton reaction – Fenton reactions creates a hydroxyl radical that is highly reactive.

Ferrous iron can cross the outer membrane through porins. It then requires a ‘permease’ complex to cross the inner membrane.

Bacterial exoproducts aid iron uptake:

-            Proteases can degrade host iron-binding proteins; released iron is bound by siderophores

-            Haemolysins lyse blood cells and promote access to host haemoproteins

-            Phenazines are redox-active metabolites that can reduce Fe3+ to Fe2+

Quorum sensing (QS):

It is a form of cell-to-cell communication. Cells will co-ordinate what they are doing. It enables single-celled organisms to participate in cooperative group behaviour. It’s characterised by the secretion and detection of small molecules. Cells will co-ordinate what they are doing. Bacteria produce many secreted factors that are important for growth/survival, or play a role in infection, individual cells cannot produce sufficient quantities to gain much benefit, however populations of bacteria can produce sufficient quantities of these factors, if they co-ordinate their behaviour.

Acyl Homoserine Lactones (AHLs):

They are a widespread auto-inducer family in Gram-neg. The molecules consist of a lactone ring and an acyl chain. They passively diffuse across the bacterial membranes. AHL binds to the LuxR family transcriptional regulator, triggering transcriptional response.

There are three different auto-inducer molecules:

-            3-oxo-C12-HSL

-            C4-HSL

-            PQS

In order to cause infection, bacteria must be able to adapt to environmental conditions in vitro. Environmental cues include:

-            Temperature

-            Acidic pH

-            Osmolarity

-            Population density

Two-component systems (TCSs):

TCSs are phospho-relay systems that facilitate adaptive responses to environmental stimuli

(Membrane spanning/inner membrane in Gram neg) sensor kinases (SK):

N-terminal ligand-binding domain linked to a C-terminal catalytic core (HK):

-            HK contains a conserved histidine, which is phosphorylated using ATP

-            Often the identity signal is unknown

It is usually transmembrane, with the sensing domain located outside of the plasma membrane

Response regulator (RR):
**** N-terminal REC domain receives the phosphate from the SK on a conserved aspartate. The output domain facilitates the response. Brings about change in gene expression.

TCSs must be tightly regulated to facilitate transient adaptive responses to stimuli. Phosphorylated forms of RRs typically have a short half life:

-            Many RRs have autophosphatase activity

-            Many SKs possess phosphatase activity

Basal activity is low unless stimulus is present.

Specificity is government by amino acids at the interface of the SK and RR pair.

EnvZ-OmpR two-component system:

Responds to changes in osmolarity. It regulated the expression of the genes encoding OmpF and OmpC porins. It controls the relative ratio of OmpF (wide diameter, low osmolarity porin):OmpC (narrow diameter, high osmolarity porin) to facilitate adaption to osmolarity.

EnvZ – SK

OmprR – RR

Under conditions of low osmolarity, EnvZ primarily exhibits phosphatase activity, keeping levels of OmpR-P low, keeping basal activity low. The available OmpR-P binds high affinity binding sites upstream of ompF, promoting its expression.

Under conditions of high osmolarity, EnvZ kinase activity is favoured, leading to high levels of OmpR-P. OmpR-P binds additional low affinity binding sites upstream of ompF that inhibit ompF expression. OmpR binds to low affinity sites upstream of ompC, that enhance ompC expression.

CheAY two-component system:

CheA is a cytoplasmic sensor kinase, which interacts with chemoreceptors via CheW. When activated CheA phosphorylates the CheY response regulator, which interacts with the FliM proteins of the flagellar motor (the motor switch – alters direction). CheZ has a role in dephosphorylating CheY~P to resent the system.

The default mode for flagella rotation is counter-clockwise (CCW). Active CheY-P diffuses through the cytoplasm and interacts with FliM. Direction of rotation is switched from counter-clockwise to clockwise (CW) – causes the cell to tumble.

Moving up a concentration gradient:

Attractant is bound to chemoreceptors, which inhibits CheA autophosphorylation. CheY is inactive so flagellar rotation remains counter-clockwise.

Moving down a concentration gradient:

As attractant levels fall, the amount of attractant bound to chemoreceptors decreases, which activates CheA autophosphorylation. CheY is activated and interacts with FliM, resulting in clockwise flagellar rotation (tumbling). CheZ dephosphorylated CheY-P to reset the system, reinstating CCW rotation.

Core RNA polymerase has low general affinity for DNA but does not recognise promotors and cannot initiate transcription. The recruitment of a sigma factor to Rpol is essential for promotor recognition and transcription initiation – complex referred to as a ‘holoenzyme’. Different sigma factors recognise different promotor sequences and confer specificity for promotor sequences.

RpoS controls the response to multiple stresses; activity can be influenced at every level. Different sigma factor -> different/alternative gene transcribed. Bacterial cell can alter the availability of different sigma factors, thereby altering set of genes being expressed. Stress can enhance/inhibit transcription and translation.

Stress responses can be controlled by regulating the availability of the corresponding sigma factor. This can be achieved by:

-            Change the rate of synthesis/degradation

-            Through actions of anti-sigma factors

Adaptor proteins direct protein substrates to proteases for degradation. RssB is an adaptor protein for RpoS; when phosphorylated, RssB (targets RpoS to protein complex) directs RpoS to the ClpXP protease. Under energy starvation, phosphorylation of RssB is reduced, so less RpoS is targeted for degradation. IraP, IraM, and IraD are anti-adaptor proteins that inhibit RssB, stabilising RpoS.