Prokaryotic Translation &

Protein Secretion System

Nisha Kalra
Mariyam Jaffar
Faiza Ali
Yusra Akbar
8th Semester
Department of Microbiology
Faculty of Life Sciences & Informatics (FLSI)
BUITEMS
 Table of Contents

Introduction
Why resistance is a concern?
Antibiotic Pressure
Resistance Acquirement Mechanisms
Mechanisms of antibiotic resistance
Conclusion
References
Introduction
The conversion of information
encoded as DNA into the synthesis
of a polypeptide chain occurs in two
Central Dogma of Life distinct phases;
• first the ‘message’ encoded in the
DNA sequence of a gene is
converted to mRNA by
transcription,
• then this directs the assembly of
a specific sequence of amino
acids during translation
Translation occurs in the form of Genetic
code
• The sequence of bases within an mRNA molecule provides coded information
that is read in groups of three nucleotides known as codons.

• The sequence of three bases in most codons specifies a particular amino acid.

• These codons are termed sense codons.

• For example,

• codon AGC specifies the amino acid serine

• codon GGG encodes the amino acid glycine

• codon AUG, which specifies methionine, is used as a start codon; it is
usually the first codon that begins a polypeptide sequence.
• The AUG codon can also be used to specify additional methionines
within the coding sequence.
• Three codons —UAA, UAG, and UGA—are used to end the process of
translation and are known as stop codons.

• They are also called termination or nonsense codons.

• An mRNA molecule also has regions that precede the start codon and
follow the stop codon.

• Because these regions do not encode a polypeptide, they are called the
• 5ʹ-untranslated region
• 3ʹ-untranslated region
• The codons in mRNA are recognized by the anticodons in transfer RNA (tRNA)

• Anticodons are three-nucleotide sequences that are complementary to codons

• tRNA molecules carry the amino acids that correspond to the codons in mRNA
• In this way, the order of codons in mRNA dictates the order of amino acids
within a polypeptide.
Timeline of Antibiotic Resistance

Figure source: Clatworthy et al. (2007)

Why resistance is a concern?

Resistant organisms lead to treatment failure

Increased mortality
Resistant bacteria may spread in Community
Low level resistance can go undetected
Added burden on healthcare costs
Threatens to return to pre-antibiotic era
Antibiotics promote
resistance
• If a patient taking a course of
antibiotic treatment does not
complete it
• Or forgets to take the doses
regularly, then resistant strains
get a chance to build up
How do drug resistant bugs arise?

Antibiotic Pressure
Figure source: evolution.berkeley.edu
 Resistance Mechanism

1.Lack target
2.Innate efflux pumps
3.Drug inactivation (Cephalosporinase in
Klebsiella)
Acquired resistance

Mutations It refers to the change in DNA structure of the gene.

Occurs at a frequency of one per ten million cells.

E.g. Mycobacterium tuberculosis, Mycobacterium lepra , MRSA.

Often mutants have reduced susceptibility

Acquired resistance

Plasmids Extra chromosomal genetic elements can replicate

independently and freely in cytoplasm.
Plasmids which carry genes resistant ( r-genes) are called R-
plasmids.
These r-genes can be readily transferred from one R-plasmid to
another plasmid or to chromosome.
Much of the drug resistance encountered in clinical practice is
plasmid mediated
Mechanisms of Resistance Gene Transfer

Transfer of r-genes from Transfer of r-genes

one bacterium to between plasmids within
another the bacterium
• Conjugation • By transposons
• Transduction • By Integrons
• Transformation
Cont…
• Transposons are sequences of DNA that
can move around different positions
within the genome of single cell
• The donor plasmid containing the
Transposons, co-integrate with acceptor
plasmid. They can replicate during
cointegration
• Both plasmids then separate and each
contains the r-gene carrying the
transposon.
 Cont…
• Integron is a large mobile DNA can
spread Multidrug resistance
• Each Integron is packed with multiple
gene casettes, each consisting of a
resistance gene attached to a small
recognition site.
• These genes encode several bacterial
functions including resistance and
virulence.
• They cannot promote self transfer

Figure source: Gillings MR. 2014. Integrons: past, present, and future. Microbiol Mol Biol Rev 78:257–277.
Mechanisms of antibiotic resistance
• Changes in outer membrane permeability
Reduced entry of drug/porin loss
Efflux pump

• Antibiotic inactivation
β lactamases
Aminoglycoside modifying enzymes (AMEs)

• Modification of target molecule

Penicillin binding protein modifications
Modifications in the peptide side chain of the peptidoglycan
Ribosomal RNA methylation
Mutations in enzymes required for DNA synthesis
 Target Site Modification

Altered Penicillin-Binding Proteins (PBPs)

Cont…
• Methicillin resistance involves the presence of the
chromosomally localized mecA gene
 The mecA gene is the part of a mobile genetic element called
Staphylococcal Cassette Chromosome mec (SCCmec)
 SCC have mecA and crr (cassette chromosome recombinase) that attaches
it to the 3ˊ end of staphylococcal chromosome
 The genetic mechanism for the transfer of these mobile elements is
undefined
 mecA gene in MRSA encodes modified penicillin binding protein 2a (PBP
2a OR PBP 2ˊ)
 PBP 2a has low affinity for β-lactams
 Figure: Mechanism of resistance

Figure source: CDC, 2006 and http://www.jci.org/cgi/content/full/114/12/1693/F1

 Target Site Modification

Modification in the Peptide Side Chain of

Peptidoglycan Layer
Ribosomal RNA Methylation
• Mutations in genes encoding the central loop of the domain
V of the 23S rRNA in the 50S ribosomal subunit are the most
frequent determinants of linezolid resistance.
• methylation of the ribosome catalyzed by an enzyme
encoded by the erm genes (erythromycin ribosomal
methylation)
• These enzymes are capable of mono- or dimethylating an
adenine residue in position A2058 of the domain V of the
23rRNA of the 50S ribosomal subunit.
• Due to this biochemical change, the binding of the
antimicrobial molecule to its target is impaired
Figure source: Munita, J. M., & Arias, C. A. (2016). Mechanisms of Antibiotic Resistance. Microbiology spectrum, 4(2), 10.1128/microbiolspec.VMBF-0016-2015. doi:10.1128/microbiolspec.VMBF-
0016-2015
Mutations in DNA Synthesis enzymes
• The most common mechanism of high-level fluoroquinolone resistance is due
to mutation in one or more of the genes that encode the primary and
secondary targets of these drugs, the type II topoisomerases (gyrA, gyrB, parC,
and parE).

• The region where mutations arise in these genes that encode fluoroquinolone
resistance is a short DNA sequence known as the quinolone resistance-
determining region (QRDR)

• Mutations in the QRDR of these genes, resulting in amino acid substitutions,

alter the target protein structure and subsequently the fluoroquinolone-
binding affinity of the enzyme, leading to drug resistance
Figure source: Munita, J. M., & Arias, C. A. (2016). Mechanisms of Antibiotic Resistance. Microbiology
spectrum, 4(2), 10.1128/microbiolspec.VMBF-0016-2015. doi:10.1128/microbiolspec.VMBF-0016-2015
 Cont…

• Alterations of porins could be

achieved by
• a shift in the type of porins
expressed
• a change in the level of porin
expression
• impairment of the porin function

Figure source: Lucía Fernández, Robert E. W. Hancock Clinical Microbiology Reviews Oct 2012, 25 (4) 661-681
 Cont…
Changes in permeability through any of these mechanisms frequently
result in low-level resistance and are often associated with other
mechanisms of resistance, such as increased expression of efflux pumps

Figure source: Sylvie Garneau-Tsodikovaa and Kristin J. Labby MedChemCommun, 2016, 7 (1) 11-27
Cont…
• Example
 Clinical isolates of K. pneumoniae recovered before and after
antimicrobial therapy
 The post-therapy isolates were found to exhibit a shift in porin expression
from OmpK35 to OmpK36 (the latter possessing a smaller channel size)
 This alteration in the type of porin expressed correlated with a 4 – 8 fold
decrease in susceptibility for a wide range of β-lactam antimicrobials
Efflux Pump
• The production of complex bacterial machineries capable to extrude a toxic
compound out of the cell can also result in antimicrobial resistance.

• These systems may be substrate-specific (for a particular antibiotic such as tet

determinants for tetracycline and mef genes for macrolides in pneumococci)

• Or with broad substrate specificity, which are usually found in MDR bacteria
Figure: Mechanism for bacterial resistance to
tetracycline (T) caused by efflux

Figure Source:Inhibitors of bacterial ribosomal actions. (2015, 02 08).

Retrieved from iKNOWLEDGE.
Figure: Representation of different types of efflux pumps in gram-positive and gram-negative bacteria

Figure source: Munita, J. M., & Arias, C. A. (2016). Mechanisms of Antibiotic Resistance. Microbiology spectrum, 4(2), 10.1128/microbiolspec.VMBF-0016-2015. doi:10.1128/microbiolspec.VMBF-
0016-2015
 Inactivation of antibiotic: β lactam

Dr.M.Malathi Health & Medicine Oct 12, 2015

 Cont…
Genes encoding for β-lactamases are generally termed bla, followed by the
name of the specific enzyme (e.g. blaKPC) and they have been found in the
chromosome or localized in MGEs as part of the accessory genome

Figure source: Dr.M.Malathi Health & Medicine Oct 12, 2015

 Inactivation of antibiotic – Aminogylocoside
modifying enzymes (AMEs)
• Resistance via AMEs that covalently
modify the hydroxyl or amino groups of
the aminoglycoside molecule
• These enzymes are usually harbored in
MGEs, but genes coding for AMEs have
also been found as part of the
chromosome in certain bacterial
species

Figure source: Insight into Acinetobacter baumannii : pathogenesis, global resistance, mechanisms of resistance, treatment options, and alternative modalities - Scienti fic Figure on
ResearchGate. Available from: https://www.researchgate.net/figure/Different-mechanisms-of-resistance-in-A-baumannii-I-beta-lactams-II_fig1_327141952 [accessed 10 Jul, 2019
Cont…
• The nomenclature to classify the multiple AMEs considers;

 Their biochemical activity

 The site of the modification (depicted by a number from 1 to 6 corresponding to the
particular carbon on the sugar ring)
 Cont…
 Aminoglycoside Acetyltransferases (AAC Family)
 Utilize intracellular acetyl-CoA as a co-substrate,
catalyzing the formation of a biologically stable amide
with the aminoglycoside

 Aminoglycoside Phosphotransferases (APH Family)

 Catalyze the phosphorylation of specific
aminoglycoside hydroxyl residues, using intracellular
ATP as a phosphate donor

 Aminoglycoside Nucleotidyltransferases (ANT Family)

 utilize the co- substrate ATP to transfer an AMP
moiety to selected aminoglycoside hydroxyl groups
Figure source: Biochemical Logic of Antibiotic Inactivation and Modification - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/nactivation-of-the-
aminoglycoside-gentamicin-C1a-by-aminoglycoside-modifying-enzymes_fig10_226609713 [accessed 10 Jul, 2019]
Cont…

• Example
 The APH family is widely distributed in gram-positive and gram-negative bacteria
and alters kanamycin and streptomycin, but spares gentamicin and tobramycin
 And, AAC(6′)-I is mainly found in gram-negative clinical isolates including
Enterobacteriaceae, Pseudomonas and Acinetobacter and affects most
aminoglycosides including amikacin and gentamicin
Conclusion
• Emergence of resistance among the most important bacterial pathogens is
recognized as a major public health threat affecting humans worldwide
• The bacterial response to the antibiotic “attack” is the prime example of
bacterial adaptation and the pinnacle of evolution
• Resistance result in mutational adaptations, acquisition of genetic
material or alteration of gene expression producing resistance to virtually
all antibiotics currently available in clinical practice
• Therefore, understanding the biochemical and genetic basis of resistance
is of paramount importance to design strategies to reduce the emergence
and spread of resistance and devise innovative therapeutic approaches
against multidrug-resistant organisms
References
• Al-mohanna, M. T. Antibiotics and chemotherapeutic agents; 2016.
• D'Costa, Vanessa & D. Wright, Gerard. (2009). Biochemical Logic of Antibiotic Inactivation and Modification.
10.1007/978-1-59745-180-2_8.
• Ega & B Dogo, E & Murtala, R & Dawakin Kudu, Abubakar. (2018). AN OVERVIEW ON METHICILLIN RESISTANT
Staphylococcus aureus.
• Garima Kapoor, Saurabh Saigal1, A. E. Action and resistance mechanisms of antibiotics: A guide for clinicians.
Journal of Anaesthesiology Clinical Pharmacology, 2017;33(3), 11.
• Geo F. Brooks, Karen C. Carroll, Janet S. Butel, Stephen A. Morse, Timothy A. Mietzner. Jawetz, Melnick, & Adelberg's
Medical Microbiology 26th edition. McGraw-Hill; 2012.
• Inhibitors of bacterial ribosomal actions. (2015, 02 08). Retrieved from iKNOWLEDGE.
• Liwa, A. C., & Jaka, H. Antimicrobial resistance: Mechanisms of action of antimicrobial agents. The Battle Against
Microbial Pathogens: Basic Science, Technological Advances and Educational Programs, 2015; 876–885.
• Munita, J. M., & Arias, C. A. (2016). Mechanisms of Antibiotic Resistance. Microbiology spectrum, 4(2),
10.1128/microbiolspec.VMBF-0016-2015. doi:10.1128/microbiolspec.VMBF-0016-2015
• Neu HC, Gootz TD. Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical
Branch at Galveston; 1996.
Thank you!
Q&A