HIV and Bacteria (Week 2)

Question 1

Essential parts of HIV virion

Answer 1

1) 2 identical ssRNA pieces (diploid)
2) Nucleocapsid (NC) proteins
3) 3 essential enzymes: protease, reverse transcriptase, integrase
4) Capsid proteins (CA) of which major one is p24 (forms icosahedral capsid)
5) Matrix proteins under envelope (like influenza)
6) Surface glycoproteins: gp120, gp41

Question 2

HIV genome

Answer 2

1) Long terminal repeats (LTRs) at two ends: sticky ends used by integrase for insertion into host DNA and promoter/enhancer once incorporated into host DNA
2) Gag: major structural proteins like nucleocapsid (NC), p24 (capsid), matrix (MA)
3) Pol: protease, integrase and reverse transcriptase
4) Env: envelope proteins that form gp120 and gp41 once glycosylated
5) Tat: viral transactivator protein that activates transcription
6) Rev: binds env gene to decrease splicing in order to REV up reading of gag, pol, and env to produce more virions (also produces vif, vpu, vpr)
7) Nef: unclear, can both positively and negatively regulate HIV expression

Question 3

Where is there a high concentration of hypervariable regions?

Answer 3

1) gp120 (within the env gene; specifically the V3 loop): this is why it is so hard to make a vaccine, because envelope constantly changing
2) Gene for reverse transcriptase: this is why RT inhibitors don’t work well either

Question 4

Progression to AIDS in 3 stages

Answer 4

1) Acute viral illness like mononucleosis (fever, lymphadenopathy, pharyngitis, etc) about 1 month after initial exposure; viremia, viruses spread to infect lymph nodes and macrophages, then HIV-specific immune response arises resulting in decreased viremia and resolution of symptoms; HIV replication still continues in lymph nodes and peripheral blood
2) Clinical latency for 8 years where no symptoms (other than maybe lymphadenopathy) but HIV continues to replicate and destroy CD4 T cells; then begin to get bacterial and skin infections and constitutional symptoms (fever, weight loss, night sweats)
3) AIDS (CD4 T cells < 200 and/or AIDS-defining opportunistic infection) for 2 years before death

Question 5

Mechanisms of T-cell death caused by HIV

Answer 5

1) When virion budding, gp160 binds adjacent CD4 receptors on same T cell and tear membrane
2) Infected cell to noninfected cell fusion via gp160 causing multinucleated giant cells (as many as 500 cells can fuse!)
3) GP160 marks T cell as “non-self” to cause autoimmune destruction by CD8 T cells

Question 6

As CD4 T cells are lost during HIV infection, what happens to other parts of immune system?

Answer 6

1) Multinucleated giant cells form and allow virus to pass from cell to cell protected from circulating antibodies
2) HIV doesn’t infect B cells but there is somehow polyclonal activation of B cells causing outpouring of immunoglobulins –> hypergammaglobulinemia causes immune complex formation and autoantibody production –> also diminished ability to produce antibodies in response to new antigens
3) HIV infects monocytes and macrophages and doesn’t kill them but (1) creates reservoir of HIV to replicate, and (2) causes carries HIV across BBB to cause brain disease

Question 7

Diseases seen in AIDS

Answer 7

Neurologic: AIDS dementia complex, aseptic meningitis

Malignancies: B cell lymphoma, Kaposi’s sarcoma

Opportunistic infections: mycobacterium tuberculosis, mycobacterium avium-intracellulare (MAI), candida albicans (thrush), cryptococcus neoformans, histoplasma capsulatum, coccidioides immitis, herpes zoster, oral hairy leukoplakia, herpes simplex, CMV (retinitis), pneumocystic carinii pneumonia (PCP), toxoplasm gondii, cryptosporidium, microsporidia, isospora belli

Question 8

Fuzeon (enfuvirtide)

Answer 8

Drug that prevents fusion of HIV and cell membrane (prevents 6-helix bundle from forming)

Question 9

Maraviroc (Selzentry)

Answer 9

Drug that binds allosterically to CCR5 to inhibit it, so can no longer bind gp120 so HIV cannot enter cell

Note: before giving this, should check to make sure patient actually has CCR5 virus (M-tropic) because this drug won’t help if patient has CXCR4 virus!

Question 10

Ibalizumab

Answer 10

Drug that binds CD4 (anti-CD4 monoclonal antibody) so HIV cannot get into cell

Question 11

Tropism of HIV

Answer 11

M-tropic: macrophages contain only CCR5; in early stage of HIV, viruses grow in macrophages

T-tropic: T cells (mature?) contain only CXCR4; in later stage of HIV, viruses grow in T cells

Note: primary T cells contain both CCR5 and CXCR4

Question 12

What does it mean if someone has a CXCR4-using HIV virus?

Answer 12

Bad prognostic sign

Doesn’t mean CXCR4-using virus is the cause of disease progression –> CXCR4-using virus may actually be LESS fit, and thus arise late in disease when immune system is damaged

Question 13

What mutations result in a person being very resistant to HIV infection?

Answer 13

delta32ccr5

Deletion 32 in the CCR5 coreceptor gene

Homozygous for deletion confers resistance to HIV infection

Heterozygous for deletion means will progress to AIDS slower

Question 14

Nucleotide reverse transcriptase inhibitors (NRTI)

Answer 14

Drugs that are nucleotide analogues that are chain terminators

Azidothymidine (AZT) and lamivudine (3TC) used together because if you become resistant to one you become susceptible to the other

Question 15

Non-nucleotide reverse transcriptase inhibitors (NNRTI)

Answer 15

Drugs that are not nucleotide analogues, but actually bind allosterically to reverse transcriptase protein to inactivate it

Question 16

RNaseH

Answer 16

Degrades RNA strand of an RNA-DNA hybrid, which is necessary as RNA –> dsDNA

Question 17

Integrase inhibitors

Answer 17

Drug that inhibits integrase (which is an enzyme that allows HIV to integrate into terminally differentiated cells and not just dividing cells like other viruses)

Ex: Raltegravir

Question 18

Protease inhibitors

Answer 18

Prevent viral assembly because can’t process structural proteins –> no virus budding

Note: probably do more than just inhibit proteases

Ex: Saquinavir

Question 19

4 steps that HIV drugs target

Answer 19

1) Entry: enfuvirtide (Fuzeon) blocks fusion, maraviroc (Selzentry) blocks CCR5
2) Reverse transcription: NRTIs and NNRTIs
3) Integrating genetic info into cell: raltegravir (Isentress)
4) Proteases allow virus to be put together and bud: protease inhibitors

Question 20

Our host cell’s intrinsic immunity that fights off HIV and the HIV mechanisms that have evolved to fight back

Answer 20

1) APOBEC3G in host cell is a cytosine deaminase that causes hypermutation to try to kill virus, but HIV has vif that destroys APOBEC3G
2) The TRIM5-alpha that humans have is bad and has one mutation that prevents it from doing its original job which was to prevent HIV uncoating (monkey TRIM5-alpha DOES prevent HIV uncoating!)
3) Tetherin in host cell prevents (enveloped) viruses from leaving the cell. HIV’s vpu gene binds and degrades tetherin

Question 21

Window period

Answer 21

Don’t yet have antibodies to HIV, but do have very high viral load

Note: usually you develop antibodies within the first few days of infection, but with HIV don’t develop antibodies until 6 weeks later

Question 22

Why is it good to treat HIV early?

Answer 22

Because you’re just dealing with a small (more homogenous) founder population

Later on, virus grows exponentially in lymphatic tissues

Semi-unrelated note: destruction of GALT early on may be key in HIV pathogenesis

Question 23

Drugs to treat HIV

Answer 23

NRTI reverse transcriptase inhibitors: AZT, 3TC

NNRTI reverse transcriptase inhibitors: nevirapine, efavirenz, delaviridine

Protease inhibitors: saquinavir, ritonavir, indinavir, nelfinavir, amprenavir

Entry inhibitors: enfuviritide, maraviroc

Integrase inhibitors: raltegravir

Question 24

Early complications of HIV infection (CD4 > 200)

Answer 24

Oral or vaginal candidiasis

Oral hairy leukoplakia

Herpes zoster (shingles)

Seborrheic dermatitis

Recurrent or atypical HSV

Kaposi’s sarcoma

Extensive, recurrent condyloma (HPV)

Diseases that have serious complications: syphilis, salmonella, campylobacter, H influenzae, Strep pneumoniae, Staph aureus, M tuberculosis, Hep C

Question 25

Late complications of HIV infection (CD4 \< 200)

Answer 25

**Pneumocystis jiroveci** Cryptococcus neoformans Toxoplasma gondii (reactivation) CMV reactivation Aspergillus infection Mycobacterium avium infection (MAI) Cryptosporidiosis Lymphomas Wasting disease

Question 26

Important adverse effect of NRTIs

Answer 26

**Lactic acidosis** **Fat redistribution** (loss of facial fat, buffalo hump)

Question 27

Bacteria

Answer 27

Microscopic, unicellular prokaryotes Multiply by **binary fission** (divide in half) Present everywhere! Human normal flora (microbiome) in skin, nose, pharynx, mouth, gut, vagina, etc contains 500-1000 diff species! 10x as many bacterial cells as human cells

Question 28

4 ways that bacteria exchange DNA (horizontal gene transfer)

Answer 28

1) **Transformation**: Naked DNA fragment from one bacterium released during cell lysis binds to cell wall of second bacterium, second bacterium takes up DNA intracellularly and incorporates it into its genome by homologous recombination 2a) **Generalized Transduction**: **virulent** bacteriophage infects one bacterium, accidentally packages bacterial DNA, exits bacterium during lysis, infects a second bacterium and injects first bacterium's DNA, which incorporates into second bacterium by homologous recombination 2b) **Specialized Transduction**: **temperate** bacteriophage infects one bacterium, incorporates its DNA into bacterial chromosome (called prophage with lysogenic bacterium), when activated, prophage DNA spliced out of bacterial chromosome but **error in splicing** brings a flanking bacterial gene (ie biotin or galactose synthesis) with it, so when infects second bacterium that DNA can incorporate via homologous recombination 3) **Conjugation**: one bacterium has an F plasmid (F+) which encodes enzymes and proteins to form sex pilus which pokes into second (F-) bacterium and one strand of DNA passed through conjugal bridge to transfer entire plasmid (now both bacteria F+) 4) **Transposons**: mobile genetic elements can insert themselves into donor chromosome (phages, plasmids, or bacterial chromosomes) without having DNA homology

Question 29

5 ways a bacterium can become antibiotic resistant

Answer 29

1) Acquire a gene that **destroys and inactivates** the antibiotic (ex: acquire gene for beta-lactamase/penicillinase which cleaves beta-lactam ring of penicillin) 2) Acquire a gene that **modifies and inactivates** the antibiotic (ex: acetylation, phosphorylation or adenylation) 3) Acquire a gene that encodes an **efflux pump** to pump the antibiotic out of the bacteria (ex: tetracycline resistance) 4) Mutation in a **porin** gene can cause decreased permeability to the antibiotic (ex: porin mutations in cephalosporin) 5) Mutate the **target** to preserve its function but not allow the antibiotic to bind/inactivate it anymore (ex: streptomycin

Question 30

Streptococcal classification based on ability to lyse RBCs

Answer 30

**Beta**-hemolytic streptococci: completely lyse RBCs (**clear**) **Alpha**-hemolytic streptococci: partially lyse RBCs (**green**) Gamma-hemolytic streptococci: unable to hemolyze RBCs (non-hemolytic)

Question 31

Streptococcal classification based on Lancefield antigens

Answer 31

Antigenic characteristis of C carbohydrate on cell wall A, B, D are the only clinically relevant pathogens Note: 2 clinically relevant species don't have Lancefield antigens (Streptococcus pneumoniae and Viridians group streptococci)

Question 32

M protein

Answer 32

Major virulence factor for **group A streptococcus** Inhibits activation of complement (C3b) and protects organism from phagocytosis (**anti-phagocytic**) However **antibodies** can bind to M protein and opsonize and then macrophages and neutrophils will destroy the organism

Question 33

Pathogenic enzymes of Beta-hemolytic group A streptococci

Answer 33

1) **Streptolysin O**: oxygen labile; destroys RBCs and WBCs; **antigenic** (develop **ASO** antibodies if suspect infection) 2) **Streptolysin S**: oxygen stable; destroys RBCs and WBCs; not antigenic 3) **Pyrogenic exotoxin** (**erythrogenic toxin**): only found in a few strains of beta-hemolytic group A strep, but when present it causes **scarlet fever**; some strains produce pyrogenic exotoxins that are superantigens and stim T cells to pour out inflammatory cytokines (streptococcal TSS) 4) **Streptokinase**: activates proteolytic enzyme plasmin 5) Others: hyaluronidase, DNAases (hydrolyzes DNA in pus; promotes local spread), anti-C5a peptidase (cleaves C5a to inhibit complement), others

Question 34

Rheumatic fever

Answer 34

Fever Myocarditis (--\> mitral valve damage) Joint swelling (arthritis) Chorea (Sydenham's chorea or St. Vitus dance) Subcutaneous nodules Erythema marginatum

Question 35

Proteins of staphylococcus aureus that stick out of microcapsule (around cell wall) to disable our immune system

Answer 35

1) **Protein A**: bind Fc portion of IgG to protect organism from opsonization and phagocytosis 2) **Coagulase**: fibrinogen --\> fibrin causes clumping (gold medalists clump together!) to protect from phagocytosis 3) Hemolysins: alpha, beta, gamma, delta; destroy RBCs, neutrophils, macrophages, platelets 4) Leukocidins: destroy WBCs (leukocytes) 5) Penicillinase: secreted form of beta-lactamase disrupts beta-lactam portion of penicillin molecule to inactivate it 6) Novel penicillin binding protein: (transpeptidase) necessary for cell wall peptidoglycan formation but is inhibited by penicillin

Question 36

Proteins of staphylococcus aureus that tunnel through tissue

Answer 36

1) **Hyaluronidase** ("spreading factor"): breaks down proteoglycans in connective tissue 2) **Staphylokinase**: lyses fibrin clots (like streptokinase) 3) **Lipase**: degrades fats and oils which accumulate on surface of our body (facilitates S aureus colonization of sebaceous glands) 4) **Protease**: destroys tissue proteins

Question 37

Exotoxins of S aureus

Answer 37

1) **Exfoliatin**: diffusible exotoxin that causes skin to slough off (**scalded skin syndrome**) 2) **Enterotoxins**: heat stable; cause **food poisoning** (vomiting and diarrhea) 3) Toxic shock syndrome toxin (**TSST-1**): analogous to pyrogenic toxin produced by group A beta-hemolytic streptococci, but more deadly; causes TSS and is found in 20% of S aureus; **superantigens** bind to MHC-II to cause outpouring of cytokines --\> TSS

Question 38

Staphylococcus aureus diseases caused by exotoxin release vs. direct organ invasion

Answer 38

**Exotoxin release**: gastroenteritis, TSS, scalded skin syndrome **Direct organ invasion**: pneumonia, meningitis, osteomyelitis, acute bacterial endocarditis, septic arthritis, skin infections, bacteremia/sepsis, UTI

Question 39

Are staphylococci resistant to penicillin?

Answer 39

**Yes**, most staphylococci secrete **penicillinase** which break down penicillin

Question 40

Methicillin-resistant Staphylococcus aureus (MRSA)

Answer 40

Acquired multi-drug resistance, even against penicillinase-resistant penicillins (methicillin, nafcillin, etc) **mecA** gene encodes a new **penicillin binding protein 2A** that **takes over the job of peptidoglycan cell wall assembly** when normal PBP is inhibited

Question 41

Groups of antibacterial agents based on what stage they act on

Answer 41

1) Inhibit synthesis/damage peptidoglycan **cell wall** (beta-lactams) 2) Inhibit synthesis/damage **cytoplasmic** **membrane** (polymyxins, daptomycin) 3) Modification of synthesis/metabolism of **nucleic acids** (quinolones, rifampin) 4) Inhibition/modification of **protein synthesis** (aminoglycosides, tetracyclines) 5) Disruption of **bacterial energetics** (metronidazole)

Question 42

Targets of antibacterial agents

Answer 42

1) **Cell wall synthesis**: penicillins, cephalosporins, vancomycin, bacitracin 2) **Cytoplasmic membrane integrity**: polymixin, daptomycin 3) **Protein synthesis**: tetracyclines, aminoglycosides, macrolides, others 4) **DNA synthesis**: ciprofloxacin and other quinolones 5) **RNA synthesis**: rifampin and other rifamycins 6) **Folate synthesis**: trimethoprim and sulfas

Question 43

Minimum inhibitory concentration (MIC)

Answer 43

Smallest amount of drug that will inhibit growth of bacteria in benchtop testing The **lower** the MIC, the more **susceptible** the pathogen to that drug

Question 44

Host factors to consider when giving antibacterial drug

Answer 44

1) Adequate **concentration** of drug delivered to **site** of infection (local conc greater than MIC) 2) Subinhibitory concentrations may still be beneficial and host can finish killing (alter bacterial adherence, morphology, aid in phagocytosis and killing) 3) **Protein** **binding** of drug can affect how much drug gets to site

Question 45

Drugs that affect developing bone and teeth

Answer 45

Tetracyclines stain teeth (even of unborn fetus!) Quinolones impair bone and cartilage growth

Question 46

Complications of antibiotic therapy

Answer 46

**Hypersensitivity reactions**: beta-lactams, sulfonamides, others **Organ toxicity**: ototoxicity/nephrotoxicity of aminoglycosides, hepatitis due to isonazid or rifampin **Superinfection**: changing normal flora can cause C. difficile colitis or Candida pharyngitis

Question 47

CA-MRSA vs. HA-MRSA

Answer 47

**CA-MRSA**: first reported in 1990s; have **PVL** virulence gene; cause furunclulosis, necrotizing pneumonia; mec IV resistance genes; **sensitive** to clindamycin, TMP-SMX, doxycycline **HA-MRSA**: first reported in 1970s; have no PVL virulence gene; cause multiple infections; mec I-III; **resistant** to clindamycin, TMP-SMX, doxycycline

Question 48

Tetanus toxins

Answer 48

1) **Tetanolysin**: related to clostridial hemolysins and streptolysin O (oxygen-labile) 2) **Tetanospasmin**: A and B chains (A internalized and moves to CNS where blocks release of NTs for inhibitory synapses)

Question 49

Capsule of Bacillus anthracis

Answer 49

Poly-D-glutamic acid capsule (different from other capsules which are polysaccharide!)

Question 50

3 anthrax exotoxins

Answer 50

1) Edema factor (EF): adenylate cyclase exotoxin increases cAMP; causes edema and impairs neutrophil function 2) Lethal factor (LF): Zinc dependent protease increases macrophage oxidative burst and proinflammatory cytokines; impairs actin polymerization and neutrophil chemotaxis 3) Protective antigen (PA): brings EF and LF together to act

Question 51

Viridans group streptococci

Answer 51

S. sanguinis, S. salivarius, S. mitis, S. mutans, etc Normal flora of oropharynx and GI tract **Alpha-hemolytic** (verde = green!) Cause **dental cavities**, subacute bacterial **endocarditis** (especially after acute rheumatic fever--damaged heart valves), **abscesses**

Question 52

Enterococci (Group D strep)

Answer 52

E. faecalis, E. faecium Normal flora in bowel Major nosicomial pathogen Cause UTI, septicemia, endocarditis Very antibiotic resistant (VRE = vancomycin resistant enterococci)

Question 53

Catalase + bacteria

Answer 53

Staph aureus Serratia marcescens Salmonella Klebsiella Burkholderia cepacia Nocardia Aspergillus Candida Note: these infect people with chronic granulomatous disease (CGD) who don't have NADPH oxidase and can't do respiratory burst to kill bacteria with H2O2 (catalase + bacteria really make it so they can't be killed because catalase breaks down any H2O2 that IS made so it won't damage them!)