Characterization of the genetic switch from phage phi13 important for Staphylococcus aureus colonization in humans

Temperate phages are bacterial viruses that either reside integrated in a bacterial genome as lysogens or enter a lytic lifecycle. Decision between lifestyles is determined by a switch involving a phage-encoded repressor, CI, and a promoter region from which lytic and lysogenic genes are divergently transcribed. Here we investigate the switch of phage phi13 from the human pathogen Staphylococcus aureus. phi13 encodes several virulence factors and is prevalent in S. aureus strains colonizing humans. We show that the phi13 switch harbors a cI gene, a predicted mor (modulator of repression) gene, and three high-affinity operator sites binding CI. To quantify the decision between lytic and lysogenic lifestyle, we introduced reporter plasmids that carry the 1.3 kb switch region from phi13 with the lytic promoter fused to lacZ into S. aureus and B. subtilis. Analysis of beta-galactosidase expression indicated that decision frequency is independent of host factors. The white “lysogenic” phenotype, which relies on expression of cI, could be switched to a stable blue “lytic” phenotype by DNA damaging agents. We have characterized lifestyle decisions of phage phi13, and our approach may be applied to other temperate phages encoding virulence factors in S. aureus.

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Phylogeny Characterization of Seb Gene Encoding Enterotoxins in Staphylococcus aureus Isolated from Raw Milk and Cheese

International Journal of Drug Delivery Technology ◽

10.25258/ijddt.v9i3.13 ◽

2019 ◽

Vol 9 (o3) ◽

Author(s):

Fatima N. Aziz ◽

Laith Abdul Hassan Mohammed-Jawad

Keyword(s):

Staphylococcus Aureus ◽

Food Poisoning ◽

Raw Milk ◽

Staphylococcal Enterotoxin B ◽

Human Pathogen ◽

Conventional Pcr ◽

Biochemical Tests ◽

Specific Primers ◽

Gene Encoding

Food poisoning due to the bacteria is a big global problem in economically and human's health. This problem refers to an illness which is due to infection or the toxin exists in nature and the food that use. Milk is considered a nutritious food because it contains proteins and vitamins. The aim of this study is to detect and phylogeny characterization of staphylococcal enterotoxin B gene (Seb). A total of 200 milk and cheese samples were screened. One hundred ten isolates of Staphylococcus aureus pre-confirmed using selective and differential media with biochemical tests. Genomic DNA was extracted from the isolates and the SEB gene detects using conventional PCR with specific primers. Three staphylococcus aureus isolates were found to be positive for Seb gene using PCR and confirmed by sequencing. Sequence homology showed variety range of identity starting from (100% to 38%). Phylogenetic tree analyses show that samples (6 and 5) are correlated with S. epidermidis. This study discovered that isolates (A6-RLQ and A5-RLQ) are significantly clustered in a group with non- human pathogen Staphylococcus agnetis.

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Optical Mapping Reveals a Large Genetic Inversion between Two Methicillin-Resistant Staphylococcus aureus Strains

Journal of Bacteriology ◽

10.1128/jb.00325-09 ◽

2009 ◽

Vol 191 (18) ◽

pp. 5717-5723 ◽

Cited By ~ 22

Author(s):

Sanjay K. Shukla ◽

Jennifer Kislow ◽

Adam Briska ◽

John Henkhaus ◽

Colin Dykes

Keyword(s):

Staphylococcus Aureus ◽

Optical Mapping ◽

Bacterial Genome ◽

Clinical Importance ◽

Restriction Map ◽

Nucleotide Polymorphisms ◽

Specific Pcr ◽

Genetic Inversion

ABSTRACT Staphylococcus aureus is a highly versatile and evolving bacterium of great clinical importance. S. aureus can evolve by acquiring single nucleotide polymorphisms and mobile genetic elements and by recombination events. Identification and location of novel genomic elements in a bacterial genome are not straightforward, unless the whole genome is sequenced. Optical mapping is a new tool that creates a high-resolution, in situ ordered restriction map of a bacterial genome. These maps can be used to determine genomic organization and perform comparative genomics to identify genomic rearrangements, such as insertions, deletions, duplications, and inversions, compared to an in silico (virtual) restriction map of a known genome sequence. Using this technology, we report here the identification, approximate location, and characterization of a genetic inversion of ∼500 kb of a DNA element between the NRS387 (USA800) and FPR3757 (USA300) strains. The presence of the inversion and location of its junction sites were confirmed by site-specific PCR and sequencing. At both the left and right junction sites in NRS387, an IS1181 element and a 73-bp sequence were identified as inverted repeats, which could explain the possible mechanism of the inversion event.

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Characterization of Virulence Factors in Enterotoxin-Producing Staphylococcus Aureus From Bulk Tank Milk

10.21203/rs.3.rs-1200960/v1 ◽

2021 ◽

Author(s):

Hye-Ri Jung ◽

Young Ju Lee

Keyword(s):

Staphylococcus Aureus ◽

Virulence Factors ◽

Virulence Genes ◽

Bovine Mastitis ◽

Bulk Tank Milk ◽

Genetic Characteristics ◽

Enterotoxin Genes ◽

High Prevalence ◽

Bulk Tank

Abstract Background: Staphylococcus aureus, a persistent and chronic mastitis-causing pathogen, produces various virulence factors, including enterotoxins. This study analyzed the genetic characteristics of bovine mastitis-related virulence factors to evaluate potential pathogenesis in S. aureus isolated from bulk tank milk.Results: Among 93 S. aureus isolates from 396 dairy farms in six factories operated by three dairy companies in Korea, 40 (43.0%) isolates carried at least one or more enterotoxin genes and there were significant differences between factories within the same company (p < 0.05). Moreover, S. aureus carrying enterotoxin genes showed a higher prevalence in all virulence genes tested in this study except for pvl and lukM, which were not detected in any isolate, than the isolates without enterotoxin genes. In particular, the prevalence of six genes (hla, hlb, lukED, fnbA, clfA, and clfB) was significantly higher in S. aureus carrying enterotoxin genes than isolates without enterotoxin genes (p < 0.05). The most common multilocus sequence type (ST) of 40 enterotoxin-producing isolates was ST188, and all isolates of ST188 harbored the see gene. However, none of the isolates of ST1 and ST72 carried the see gene, and all isolates of ST1 carried the seh gene.Conclusions: Although S. aureus isolated from bulk tank milk, not from mastitis, had a high prevalence of enterotoxins and virulence factors simultaneously, posing a public health threat. Moreover, high enterotoxins in bulk tank milk may be reflected by poor hygiene; therefore, it is important to develop strong monitoring and sanitation programs to ensure that dairy factories produce hygienic milk.

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Prevalence of Agr phase variants in Staphylococcus aureus

Access Microbiology ◽

10.1099/acmi.ac2020.po0002 ◽

2020 ◽

Vol 2 (7A) ◽

Author(s):

Vishal Gor ◽

Mitsuaki Hoshi ◽

Aya Takemura ◽

Masato Higashide ◽

Veronica Romero ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Quorum Sensing ◽

Virulence Factors ◽

Human Pathogen ◽

Accessory Gene ◽

Accessory Gene Regulator ◽

Inversion Event ◽

First Time ◽

Phase Variants ◽

Phenotypic Reversion

Staphylococcus aureus is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing Accessory Gene Regulator (Agr) system. However, a large proportion of clinical S. aureus isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr negative strains and screening for phenotypic reversion of haemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative MRSA isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. To assess the significance of our findings we screened a series of primary clinical isolates, which had undergone minimal handling post-isolation, and successfully identified a fraction which were Agr phase variants. Taken together, we propose a model where some Agr-negative S. aureus strains are phase variants who can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress.

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Staphylococcus aureus Virulence Factors Identified by Using a High-Throughput Caenorhabditis elegans-Killing Model

Infection and Immunity ◽

10.1128/iai.73.2.872-877.2005 ◽

2005 ◽

Vol 73 (2) ◽

pp. 872-877 ◽

Cited By ~ 55

Author(s):

Jakob Begun ◽

Costi D. Sifri ◽

Samuel Goldman ◽

Stephen B. Calderwood ◽

Frederick M. Ausubel

Keyword(s):

Staphylococcus Aureus ◽

Caenorhabditis Elegans ◽

Dna Replication ◽

Virulence Factors ◽

High Throughput ◽

Human Pathogen ◽

Renal Abscess ◽

C Elegans ◽

Transposon Insertion ◽

Strain Nctc

ABSTRACT Staphylococcus aureus is an important human pathogen that is also able to kill the model nematode Caenorhabditis elegans. We constructed a 2,950-member Tn917 transposon insertion library in S. aureus strain NCTC 8325. Twenty-one of these insertions exhibited attenuated C. elegans killing, and of these, 12 contained insertions in different genes or chromosomal locations. Ten of these 12 insertions showed attenuated killing phenotypes when transduced into two different S. aureus strains, and 5 of the 10 mutants correspond to genes that have not been previously identified in signature-tagged mutagenesis studies. These latter five mutants were tested in a murine renal abscess model, and one mutant harboring an insertion in nagD exhibited attenuated virulence. Interestingly, Tn917 was shown to have a very strong bias for insertions near the terminus of DNA replication.

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Characterization of the genetic switch from phage ɸ13 important for Staphylococcus aureus colonization in humans

MicrobiologyOpen ◽

10.1002/mbo3.1245 ◽

2021 ◽

Vol 10 (5) ◽

Author(s):

Camilla S. Kristensen ◽

Anders K. Varming ◽

Helena A. K. Leinweber ◽

Karin Hammer ◽

Leila Lo Leggio ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Genetic Switch

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Structural and functional characterization of SplA, an exclusively specific protease of Staphylococcus aureus

Biochemical Journal ◽

10.1042/bj20081351 ◽

2009 ◽

Vol 419 (3) ◽

pp. 555-564 ◽

Cited By ~ 24

Author(s):

Justyna Stec-Niemczyk ◽

Katarzyna Pustelny ◽

Magdalena Kisielewska ◽

Michal Bista ◽

Kevin T. Boulware ◽

...

Keyword(s):

Crystal Structure ◽

Staphylococcus Aureus ◽

Virulence Factors ◽

Expression System ◽

Functional Characterization ◽

Physiological Role ◽

Peptide Bond ◽

Amino Acid Residues ◽

Specific Protease

Staphylococcus aureus is a dangerous human pathogen whose antibiotic resistance is steadily increasing and no efficient vaccine is as yet available. This serious threat drives extensive studies on staphylococcal physiology and pathogenicity pathways, especially virulence factors. Spl (serine protease-like) proteins encoded by an operon containing up to six genes are a good example of poorly characterized secreted proteins probably involved in virulence. In the present study, we describe an efficient heterologous expression system for SplA and detailed biochemical and structural characterization of the recombinant SplA protease. The enzyme shares a significant sequence homology to V8 protease and epidermolytic toxins which are well documented staphylococcal virulence factors. SplA has a very narrow substrate specificity apparently imposed by the precise recognition of three amino acid residues positioned N-terminal to the hydrolysed peptide bond. To explain determinants of this extended specificity we resolve the crystal structure of SplA and define the consensus model of substrate binding. Furthermore we demonstrate that artificial N-terminal elongation of mature SplA mimicking a naturally present signal peptide abolishes enzymatic activity. The probable physiological role of the process is discussed. Of interest, even though precise N-terminal trimming is a common regulatory mechanism among S1 family enzymes, the crystal structure of SplA reveals novel significantly different mechanistic details.

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